Lipid formulations for delivery of messenger rna

ABSTRACT

The present invention provides, among other things, methods of delivering mRNA in vivo, including administering to a subject in need of delivery a composition comprising an mRNA encoding a protein, encapsulated within a liposome such that the administering of the composition results in the expression of the protein encoded by the mRNA in vivo, wherein the liposome comprises a cationic lipid of formula I-c: 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable salt thereof.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 61/894,299, filed Oct. 22, 2013 and U.S. Provisional ApplicationSer. No. 61/953,516, filed Mar. 14, 2014, the disclosures of which arehereby incorporated by reference.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “2006685-0686_SL.txt” onOct. 22, 2014). The .txt file was generated on Oct. 22, 2014 and is33,306 bytes in size. The entire contents of the Sequence Listing areherein incorporated by reference.

BACKGROUND

Delivery of nucleic acids has been explored extensively as a potentialtherapeutic option for certain disease states. In particular, RNAinterference (RNAi) has been the subject of significant research andclinical development. While RNAi, such as short interfering RNA (siRNA),may have therapeutic potential, it is of little use in treating diseasesinvolving deficiency of one or more proteins. messenger RNA (mRNA)therapy has become an increasingly important option for treatment ofvarious diseases, in particular, for those associated with deficiency ofone or more proteins.

SUMMARY OF THE INVENTION

The present invention provides improved methods and compositions forhighly efficient delivery and expression of mRNA and encoded protein invivo. The invention is based, in part, on the surprising discovery thatliposomes based on a particular class of cationic lipids, such as, thosehaving a structure of formula I-c described herein, are unexpectedlyeffective in delivering mRNA and producing encoded protein in vivo, moreeffective even as compared to those cationic lipids that were consideredto be among the best in delivering mRNA in the prior art. Indeed, priorto the present invention, cationic lipids have been extensively exploredas an important component of liposomes typically used to encapsulatenucleic acids including mRNA for in vivo delivery. Due to the uniquelyfragile and long structure of mRNA and the complicated in vivotranslation process, cationic lipids used in the liposomes typicallyplay two roles. First, cationic lipids promote interaction withnegatively charged mRNA during encapsulation, circulation andendocytosis, thereby capturing and protecting the mRNA. Then, onceinside cytosol, cationic lipids need to be able to release the mRNA sothat the mRNA can be translated to produce encoded protein. Somecationic lipids, in particular, those known as titratable cationiclipids are particularly effective in delivering mRNA. One example ofsuch cationic lipids known to be capable of efficient delivery of mRNAis C12-200. Surprisingly, the present inventors found that cationiclipids described herein can be even more effective in delivering variousmRNA in vivo, than those best known in the prior art including C12-200.For example, as shown in the Examples below, liposome particlesincorporating a cationic lipid described herein (e.g., cKK-E12) resultedin close to 50% higher protein expression of human Factor IX proteindetected in the plasma of administered mice, as compared toC12-200-based liposome particles. Furthermore, the plasma residence timeof different proteins expressed from mRNA delivered by cKK-E12 basedliposomes is sustained up to 7 days or longer post a singleadministration. Thus, the present inventors have demonstrated that thisclass of cationic lipids having a structure of formula I-c describedherein (e.g., cKK-E12) can be uniquely useful in delivering mRNA forhighly efficient and sustained production of protein (e.g., therapeuticprotein) in vivo. The present invention therefore permits an improvedmRNA therapy that can significantly reduce required amount of mRNA andassociated lipids, administration frequency, and possible side effects,providing more potent, safer, and patient friendly mRNA therapy forvarious diseases.

In one aspect, the present invention provides methods of deliveringmessenger RNA (mRNA) in vivo, including administering to a subject inneed of delivery a composition comprising an mRNA encoding a protein,encapsulated within a liposome such that the administering of thecomposition results in the expression of the protein encoded by the mRNAin vivo, wherein the liposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof,wherein:

p is an integer of between 1 and 9, inclusive;

each instance of R² is independently hydrogen or optionally substitutedC₁₋₆ alkyl;

each instance of R⁶ and R⁷ is independently a group of the formula (i),(ii), or (iii);

Formulae (i), (ii), and (iii) are:

wherein:

-   -   each instance of R′ is independently hydrogen or optionally        substituted alkyl;    -   X is O, S, or NR^(X), wherein R^(X) is hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted carbocyclyl,        optionally substituted heterocyclyl, optionally substituted        aryl, optionally substituted heteroaryl, or a nitrogen        protecting group;    -   Y is O, S, or NR^(Y), wherein R^(Y) is hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted carbocyclyl,        optionally substituted heterocyclyl, optionally substituted        aryl, optionally substituted heteroaryl, or a nitrogen        protecting group;    -   R^(P) is hydrogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted carbocyclyl, optionally substituted heterocyclyl,        optionally substituted aryl, optionally substituted heteroaryl,        an oxygen protecting group when attached to an oxygen atom, a        sulfur protecting group when attached to a sulfur atom, or a        nitrogen protecting group when attached to a nitrogen atom; and    -   R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally        substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl,        optionally substituted heteroC₁₋₅₀alkyl, optionally substituted        heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl,        or a polymer.

In another aspect, the present invention provides methods of treating adisease or disorder including administering to subject in need oftreatment a composition comprising an mRNA encoding a therapeuticprotein encapsulated within a liposome such that the administering ofthe composition results in the expression of the protein encoded by themRNA in one or more tissues affected by the disease or disorder, whereinthe liposome comprises a cationic lipid having a structure of formulaI-c.

In another aspect, the present invention provides compositions fordelivery of messenger RNA (mRNA) comprising an mRNA encoding a proteinencapsulated within a liposome, wherein the liposome comprises acationic lipid having a structure of formula I-c.

In some embodiments, a suitable cationic lipid is cKK-E12:

In some embodiments, a suitable liposome further comprises one or morenon-cationic lipids, one or more cholesterol-based lipids and/or one ormore PEG-modified lipids. In some embodiments, the one or morenon-cationic lipids are selected from distearoylphosphatidylcholine(DSPC), dioleoylphosphatidylcholine (DOPC),dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol(DOPG), dipalmitoylphosphatidylglycerol (DPPG),dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, a suitable liposome further comprises one or morecholesterol-based lipids. In some embodiments, the one or morecholesterol-based lipids are selected from cholesterol, PEGylatedcholesterol and DC-Chol (N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine.

In some embodiments, a suitable liposome further comprises one or morePEG-modified lipids. In some embodiments, the one or more PEG-modifiedlipids comprise a poly(ethylene) glycol chain of up to 5 kDa in lengthcovalently attached to a lipid with alkyl chain(s) of C₆-C₂₀ length. Insome embodiments, a PEG-modified lipid is a derivatized ceramide such asN-Octanoyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol)-2000].In some embodiments, a PEG-modified or PEGylated lipid is PEGylatedcholesterol or Dimyristoylglycerol (DMG)-PEG-2K.

In some embodiments, a suitable liposome comprises cKK-E12, DOPE,cholesterol and DMG-PEG2K.

In some embodiments, the cationic lipid (e.g., cKK-E12) constitutesabout 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about35-45%, or about 35-40%) of the liposome by molar ratio. In someembodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%,about 35%, about 40%, about 45%, or about 50% of the liposome by molarratio.

In particular embodiments, the ratio ofcKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:20:10 by molarratio. In particular embodiments, the ratio ofcKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:30:25:5 by molarratio. In particular embodiments, the ratio ofcKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 by molarratio.

In some embodiments, a suitable liposome has a size of or less thanabout 500 nm, 450 nm, 400 nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm,125 nm, 110 nm, 100 nm, 95 nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm,60 nm, 55 nm, or 50 nm.

In some embodiments, a composition according to the invention isadministered intravenously. In some embodiments, a composition accordingto the invention is administered via pulmonary delivery. In someembodiments, the pulmonary delivery is by aerosolization, inhalation,nebulization or instillation. In some embodiments, a compositionaccording to the invention is administered intrathecally. In someembodiments, the composition is formulated as respirable particles,nebulizable lipid, or inhalable dry powder.

In some embodiments, the expression of the protein encoded by the mRNAis detectable in liver, kidney, heart, spleen, serum, brain, skeletalmuscle, lymph nodes, skin, and/or cerebrospinal fluid.

In some embodiments, the expression of the protein encoded by the mRNAis detectable 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72 hoursafter the administration. In some embodiments, the expression of theprotein encoded by the mRNA is detectable 1 day, 2 days, 3 days, 4 days,5 days, 6 days, and/or 7 days after the administration. In someembodiments, the expression of the protein encoded by the mRNA isdetectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks after theadministration. In some embodiments, the expression of the proteinencoded by the mRNA is detectable after a month after theadministration.

In some embodiments, the protein encoded by the mRNA is a cytosolicprotein. In some embodiments, the protein encoded by the mRNA is asecreted protein. In some embodiments, the protein encoded by the mRNAis an enzyme. In some embodiments, the mRNA has a length of or greaterthan about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5kb, or 5 kb. In some embodiments, the protein encoded by the mRNA isArgininosuccinate Synthetase (ASS1), Factor IX, survival of motor neuron1, or phenylalanine hydroxylase.

In some embodiments, the mRNA is administered at a dose ranging fromabout 0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0,0.1-3.5, 0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3,0.3-5.0, 0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5,0.3-1.0, 0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5,0.5-2.0, 0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, themRNA is administered at a dose of or less than about 5.0, 4.5, 4.0, 3.5,3.0, 2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg bodyweight.

In some embodiments, the mRNA comprises one or more modifiednucleotides. In some embodiments, the one or more modified nucleotidescomprise pseudouridine, N-1-methyl-pseudouridine, 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and/or 2-thiocytidine. In someembodiments, the mRNA is unmodified.

Other features, objects, and advantages of the present invention areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the presentinvention, are given by way of illustration only, not limitation.Various changes and modifications within the scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWING

The drawings are for illustration purposes only not for limitation.

FIG. 1 shows an exemplary graph of the levels of human factor IX (FIX)detected in the serum of treated mice 24 hours after administration ofC12-200 or cKK-E12 liposomes containing FIX mRNA.

FIG. 2 shows an exemplary graph of FIX detected in the plasma of micetreated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of one of two ratios ofFIX mRNA containing cKK-E12 liposomes either 6 or 24 hours afteradministration.

FIG. 3 shows an exemplary graph of the level of ASS1 protein detected inthe livers of mice treated with 0.1, 0.3, 0.6, 1.0, or 3.0 mg/kg of ASS1mRNA-containing cKK-E12 liposomes 24 hours after administration.

FIG. 4 shows exemplary western blot analyses of ASS1 protein levels inthe liver 24 hours post administration of 0.1, 0.3, 0.6, 1.0, or 3.0mg/kg of cKK-E12 liposomes containing ASS1 mRNA.

FIG. 5 shows an exemplary graph of ASS1 protein levels in the liver ofmice 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of ASS1mRNA containing cKK-E12 liposomes (1 mg/kg). Also shown is the level ofASS1 protein 7 days after administration.

FIG. 6 shows exemplary western blot analyses of ASS1 protein levels inthe liver 0.5, 3, 6, 12, 24, 48, 72 hours after a single IV injection of1 mg/kg ASS1 mRNA containing cKK-E12 liposomes. Also shown is the levelof ASS1 protein 7 days after administration.

FIG. 7—shows detection of human ASS1 messenger RNA via in situhybridization in the livers of treated mice. Exogenous mRNA isobservable for at least 72 hr post-administration after a single dose(1.0 mg/kg) of ASS1 mRNA-loaded MD1-based lipid nanoparticles. HumanASS1 mRNA is detectable in sinusoidal cells as well as hepatocytes.

FIG. 8—shows exemplary immunohistochemical staining of ASS1 proteinlevels in mouse liver 24 hours after administration of 1 mg/kg ASS1 mRNAcontaining cKK-E12 lipid nanoparticles. Human ASS1 protein is detectablein sinusoidal cells as well as hepatocytes.

FIG. 9 shows low magnification (4×) immunohistochemical staining of ASS1protein levels in mouse liver 24 hours after administration of 1 mg/kgASS1 mRNA containing cKK-E12 liposomes. A comparison to untreated mouseliver (left) demonstrates the widespread distribution of human ASS1protein throughout the liver.

FIG. 10 shows exemplary results illustrating that cKK-E12 lipidnanoparticles efficiently delivered FL mRNA via nebulization. Mice wereexposed to milligram of encapsulated FL mRNA and analysis was performed24 hours post-exposure.

FIG. 11 illustrates detection via western blot of human SMN-1 proteinderived from exogenous hSMN-1 mRNA that was transfected into BHK-21cells. Various antibodies specific to human SMN were employed: (A)anti-SMN 4F11 antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMNantibody at 1:10,000 dilution; and (C) LSBio C138149 a-SMN antibody at1:10,000 dilution.

FIG. 12A-C illustrates multiplex nucleic acid in situ detection of humanSurvival of Motor Neuron (hSMN-1) mRNA in (A) Cervical, (B) Thoracic and(C) Lumbar spinal tissue, 24 hours post intrathecal delivery.

FIG. 13 illustrates positive detection of human SMN-1 protein producedin the spinal cord of a rat 24 hours post-intrathecal administration ofhuman SMN-1 mRNA-loaded lipid nanoparticles. Anti-human SMN 4F11antibody was employed at 1:2500 dilution. Panel A represents treated ratspinal cord tissue and panel B represents untreated rat spinal cordtissue.

FIG. 14 In vivo transfection of CFTR knockout mice with C-terminal His₁₀tagged (SEQ ID NO: 11) codon-optimized human CFTR mRNA encapsulatedwithin either a lipid (cKK-E12) or polymeric (PEI) nanoparticleformulation. Following nebulized delivery of each respective mRNAformulation, Right and Left lung tissue lysate was collected andanalyzed for CFTR expression by Western blot using anti-His antibody1187. Control CFTR knockout lung tissue and CFTR-His₁₀ HEK293 lysate(“His₁₀” disclosed as SEQ ID NO: 11) was used as a negative and positivecontrols respectively.

FIG. 15 illustrates positive detection of active firefly luciferase(FFL) protein in a treated pig lung via luminescence upon exposure toFFL/CO-CFTR-C-His₁₀ mRNA (“His₁₀” disclosed as SEQ ID NO: 11)encapsulated cKK-E12 lipid nanoparticles. Pigs were treated with 1 mgFFL+9 mg CO-CFTR-C-His₁₀ mRNA (“His₁₀” disclosed as SEQ ID NO: 11)encapsulated lipid nanoparticles via nebulization using a Pari jetnebulizer and sacrificed 24 hours post-treatment. FFL luminescence wasvisualized using an IVIS bioluminometer.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification. The publications and other reference materials referencedherein to describe the background of the invention and to provideadditional detail regarding its practice are hereby incorporated byreference.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H_(E)N—C(H)(R)—COHO. In some embodiments, an amino acid is anaturally occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a d-aminoacid; in some embodiments, an amino acid is an 1-amino acid. “Standardamino acid” refers to any of the twenty standard 1-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.As used herein, “synthetic amino acid” encompasses chemically modifiedamino acids, including but not limited to salts, amino acid derivatives(such as amides), and/or substitutions. Amino acids, including carboxyl-and/or amino-terminal amino acids in peptides, can be modified bymethylation, amidation, acetylation, protecting groups, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity. Aminoacids may participate in a disulfide bond. Amino acids may comprise oneor posttranslational modifications, such as association with one or morechemical entities (e.g., methyl groups, acetate groups, acetyl groups,phosphate groups, formyl moieties, isoprenoid groups, sulfate groups,polyethylene glycol moieties, lipid moieties, carbohydrate moieties,biotin moieties, etc.). The term “amino acid” is used interchangeablywith “amino acid residue,” and may refer to a free amino acid and/or toan amino acid residue of a peptide. It will be apparent from the contextin which the term is used whether it refers to a free amino acid or aresidue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Delivery: As used herein, the term “delivery” encompasses both local andsystemic delivery. For example, delivery of mRNA encompasses situationsin which an mRNA is delivered to a target tissue and the encoded proteinis expressed and retained within the target tissue (also referred to as“local distribution” or “local delivery”), and situations in which anmRNA is delivered to a target tissue and the encoded protein isexpressed and secreted into patient's circulation system (e.g., serum)and systematically distributed and taken up by other tissues (alsoreferred to as “systemic distribution” or “systemic delivery).

Expression: As used herein, “expression” of a nucleic acid sequencerefers to translation of an mRNA into a polypeptide, assemble multiplepolypeptides (e.g., heavy chain or light chain of antibody) into anintact protein (e.g., antibody) and/or post-translational modificationof a polypeptide or fully assembled protein (e.g., antibody). In thisapplication, the terms “expression” and “production,” and grammaticalequivalent, are used inter-changeably.

Functional: As used herein, a “functional” biological molecule is abiological molecule in a form in which it exhibits a property and/oractivity by which it is characterized.

Half-life: As used herein, the term “half-life” is the time required fora quantity such as nucleic acid or protein concentration or activity tofall to half of its value as measured at the beginning of a time period.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control subject (or multiple control subject) inthe absence of the treatment described herein. A “control subject” is asubject afflicted with the same form of disease as the subject beingtreated, who is about the same age as the subject being treated.

In Vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In Vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, about 99%, or more than about 99% of the other componentswith which they were initially associated. In some embodiments, isolatedagents are about 80%, about 85%, about 90%, about 91%, about 92%, about93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%,or more than about 99% pure. As used herein, a substance is “pure” if itis substantially free of other components. As used herein, calculationof percent purity of isolated substances and/or entities should notinclude excipients (e.g., buffer, solvent, water, etc.).

Local distribution or delivery: As used herein, the terms “localdistribution,” “local delivery,” or grammatical equivalent, refer totissue specific delivery or distribution. Typically, local distributionor delivery requires a protein (e.g., enzyme) encoded by mRNAs betranslated and expressed intracellularly or with limited secretion thatavoids entering the patient's circulation system.

messenger RNA (mRNA): As used herein, the term “messenger RNA (mRNA)”refers to a polynucleotide that encodes at least one polypeptide. mRNAas used herein encompasses both modified and unmodified RNA. mRNA maycontain one or more coding and non-coding regions. mRNA can be purifiedfrom natural sources, produced using recombinant expression systems andoptionally purified, chemically synthesized, etc. Where appropriate,e.g., in the case of chemically synthesized molecules, mRNA can comprisenucleoside analogs such as analogs having chemically modified bases orsugars, backbone modifications, etc. An mRNA sequence is presented inthe 5′ to 3′ direction unless otherwise indicated. In some embodiments,an mRNA is or comprises natural nucleosides (e.g., adenosine, guanosine,cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine,2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine,5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine,2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine,2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine); chemicallymodified bases; biologically modified bases (e.g., methylated bases);intercalated bases; modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose); and/or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

In some embodiments, the mRNA comprises one or more nonstandardnucleotide residues. The nonstandard nucleotide residues may include,e.g., 5-methyl-cytidine (“5mC”), pseudouridine (“ψU”), and/or2-thio-uridine (“2sU”). See, e.g., U.S. Pat. No. 8,278,036 orWO2011012316 for a discussion of such residues and their incorporationinto mRNA. The mRNA may be RNA, which is defined as RNA in which 25% ofU residues are 2-thio-uridine and 25% of C residues are5-methylcytidine. Teachings for the use of RNA are disclosed US PatentPublication US20120195936 and internation publication WO2011012316, bothof which are hereby incorporated by reference in their entirety. Thepresence of nonstandard nucleotide residues may render an mRNA morestable and/or less immunogenic than a control mRNA with the samesequence but containing only standard residues. In further embodiments,the mRNA may comprise one or more nonstandard nucleotide residues chosenfrom isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine and2-chloro-6-aminopurine cytosine, as well as combinations of thesemodifications and other nucleobase modifications. Certain embodimentsmay further include additional modifications to the furanose ring ornucleobase. Additional modifications may include, for example, sugarmodifications or substitutions (e.g., one or more of a 2′-O-alkylmodification, a locked nucleic acid (LNA)). In some embodiments, theRNAs may be complexed or hybridized with additional polynucleotidesand/or peptide polynucleotides (PNA). In embodiments where the sugarmodification is a 2′-O-alkyl modification, such modification mayinclude, but are not limited to a 2′-deoxy-2′-fluoro modification, a2′-O-methyl modification, a 2′-O-methoxyethyl modification and a2′-deoxy modification. In certain embodiments, any of thesemodifications may be present in 0-100% of the nucleotides—for example,more than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of theconstituent nucleotides individually or in combination.

Nucleic acid: As used herein, the term “nucleic acid,” in its broadestsense, refers to any compound and/or substance that is or can beincorporated into a polynucleotide chain. In some embodiments, a nucleicacid is a compound and/or substance that is or can be incorporated intoa polynucleotide chain via a phosphodiester linkage. In someembodiments, “nucleic acid” refers to individual nucleic acid residues(e.g., nucleotides and/or nucleosides). In some embodiments, “nucleicacid” refers to a polynucleotide chain comprising individual nucleicacid residues. In some embodiments, “nucleic acid” encompasses RNA aswell as single and/or double-stranded DNA and/or cDNA.

Patient: As used herein, the term “patient” or “subject” refers to anyorganism to which a provided composition may be administered, e.g., forexperimental, diagnostic, prophylactic, cosmetic, and/or therapeuticpurposes. Typical patients include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and/or humans). In some embodiments,a patient is a human. A human includes pre and post natal forms.

Pharmaceutically acceptable: The term “pharmaceutically acceptable” asused herein, refers to substances that, within the scope of soundmedical judgment, are suitable for use in contact with the tissues ofhuman beings and animals without excessive toxicity, irritation,allergic response, or other problem or complication, commensurate with areasonable benefit/risk ratio.

Systemic distribution or delivery: As used herein, the terms “systemicdistribution,” “systemic delivery,” or grammatical equivalent, refer toa delivery or distribution mechanism or approach that affect the entirebody or an entire organism. Typically, systemic distribution or deliveryis accomplished via body's circulation system, e.g., blood stream.Compared to the definition of “local distribution or delivery.”

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre- and post-natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Target tissues: As used herein, the term “target tissues” refers to anytissue that is affected by a disease to be treated. In some embodiments,target tissues include those tissues that display disease-associatedpathology, symptom, or feature.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of, reduce severityof and/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION

The present invention provides, among other things, methods andcompositions for delivering mRNA in vivo using improved liposomesincorporating cationic lipids described herein.

Liposomes for mRNA Delivery

As used herein, the term “liposome” refers to any lamellar,multilamellar, or solid lipid nanoparticle vesicle. Typically, aliposome as used herein can be formed by mixing one or more lipids or bymixing one or more lipids and polymer(s). Thus, the term “liposome” asused herein encompasses both lipid and polymer based nanoparticles. Inparticular, a liposome according to the present invention incorporates acationic lipid described herein. As a non-limiting example, a cationiclipid suitable for the present invention is cKK-E12, or(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione), asdescribed in more detail below. A suitable liposome may also containsecond or additional cationic lipids, helper lipids (e.g., non-cationiclipids and/or cholesterol-based lipids), PEG-modified lipids, and/orpolymers.

In some embodiments, cationic lipid(s) (e.g., cKK-E12) constitute(s)about 30-50% (e.g., about 30-45%, about 30-40%, about 35-50%, about35-45%, or about 35-40%) of the liposome by molar ratio. In someembodiments, the cationic lipid (e.g., cKK-E12) constitutes about 30%,about 35%, about 40%, about 45%, or about 50% of the liposome by molarratio.

Cationic Lipids

In some embodiments, provided liposomes or compositions providedcomprise a cationic lipid according to formula I:

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   p is an integer of between 1 and 9, inclusive;    -   each instance of Q is independently O, S, or NR^(Q);    -   R^(Q) is hydrogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted carbocyclyl, optionally substituted heterocyclyl,        optionally substituted aryl, optionally substituted heteroaryl,        a nitrogen protecting group, or a group of the formula (i), (ii)        or (iii);    -   each instance of R¹ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted carbocyclyl,        optionally substituted heterocyclyl, optionally substituted        aryl, optionally substituted heteroaryl, halogen, —OR^(A1),        —N(R^(A1))₂, —SR^(A1), or a group of formula (iv):

-   -   L is an optionally substituted alkylene, optionally substituted        alkenylene, optionally substituted alkynylene, optionally        substituted heteroalkylene, optionally substituted        heteroalkenylene, optionally substituted heteroalkynylene,        optionally substituted carbocyclylene, optionally substituted        heterocyclylene, optionally substituted arylene, or optionally        substituted heteroarylene, or combination thereof, and    -   each of R⁶ and R⁷ is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted carbocyclyl,        optionally substituted heterocyclyl, optionally substituted        aryl, optionally substituted heteroaryl, a nitrogen protecting        group, or a group of formula (i), (ii) or (iii);    -   each occurrence of R^(A1) is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted carbocyclyl,        optionally substituted heterocyclyl, optionally substituted        aryl, optionally substituted heteroaryl, an oxygen protecting        group when attached to an oxygen atom, a sulfur protecting group        when attached to an sulfur atom, a nitrogen protecting group        when attached to a nitrogen atom, or two R^(A1) groups, together        with the nitrogen atom to which they are attached, are joined to        form an optionally substituted heterocyclic or optionally        substituted heteroaryl ring;    -   each instance of R² is independently hydrogen, optionally        substituted alkyl, optionally substituted alkenyl, optionally        substituted alkynyl, optionally substituted carbocyclyl,        optionally substituted heterocyclyl, optionally substituted        aryl, optionally substituted heteroaryl, a nitrogen protecting        group, or a group of the formula (i), (ii), or (iii);    -   Formulae (i), (ii), and (iii) are:

-   -   each instance of R′ is independently hydrogen or optionally        substituted alkyl;    -   X is O, S, or NR^(X);    -   R^(X) is hydrogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted carbocyclyl, optionally substituted heterocyclyl,        optionally substituted aryl, optionally substituted heteroaryl,        or a nitrogen protecting group;    -   Y is O, S, or NR^(Y);    -   R^(Y) is hydrogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted carbocyclyl, optionally substituted heterocyclyl,        optionally substituted aryl, optionally substituted heteroaryl,        or a nitrogen protecting group;    -   R^(P) is hydrogen, optionally substituted alkyl, optionally        substituted alkenyl, optionally substituted alkynyl, optionally        substituted carbocyclyl, optionally substituted heterocyclyl,        optionally substituted aryl, optionally substituted heteroaryl,        an oxygen protecting group when attached to an oxygen atom, a        sulfur protecting group when attached to a sulfur atom, or a        nitrogen protecting group when attached to a nitrogen atom;    -   R^(L) is optionally substituted C₁₋₅₀ alkyl, optionally        substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀ alkynyl,        optionally substituted heteroC₁₋₅₀ alkyl, optionally substituted        heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀ alkynyl,        or a polymer;    -   provided that at least one instance of R^(Q), R², R⁶, or R⁷ is a        group of the formula (i), (ii), or (iii).

In some embodiments, a cationic lipid in a provided composition ormethod is a compound of formula I. In some embodiments, a cationic lipidin a provided composition or method is a compound of formula I, whereinthe compound comprises one or more basic groups. In some embodiments, acationic lipid in a provided composition or method is a compound offormula I, wherein the compound comprises one or more amino groups.

In certain embodiments, a group of formula (i) represents a group offormula (i-a) or a group of formula (i-b):

wherein each variable is independently as defined above and describedherein. In some embodiments, a group of formula (i) is a group offormula (i-a). In some embodiments, a group of formula (i) is a group offormula (i-b).

In some embodiments, at least one instance of R¹ is a group of formula(iv). In some embodiments, at least one instance of R¹ is a group offormula (iv), wherein at least one of R⁶ and R⁷ is a group of formula(i), (ii) or (iii). In some embodiments, at least one instance of R¹ isa group of formula (iv), wherein each of R⁶ and R⁷ is independently agroup of formula (i), (ii) or (iii).

In some embodiments, each R¹ is independently a group of formula (iv).In some embodiments, each R¹ is independently a group of formula (iv),wherein at least one of R⁶ and R⁷ is a group of formula (i), (ii) or(iii). In some embodiments, each R¹ is independently a group of formula(iv), wherein each of R⁶ and R⁷ is independently a group of formula (i),(ii) or (iii). In some embodiments, each R¹ is independently a group offormula (iv), wherein each of R⁶ and R⁷ is independently a group offormula (i). In some embodiments, each R¹ is independently a group offormula (iv), wherein each of R⁶ and R⁷ is independently a group offormula (ii). In some embodiments, each R¹ is independently a group offormula (iv), wherein each of R⁶ and R⁷ is independently a group offormula (iii). In some embodiments, each R¹ is independently a group offormula (iv), wherein each of R⁶ and R⁷ is independently a group offormula (i-a). In some embodiments, each R¹ is independently a group offormula (iv), wherein each of R⁶ and R⁷ is independently a group offormula (i-b).

In some embodiments, each instance of R′ is hydrogen.

In some embodiments, L is an optionally substituted alkylene.

In some embodiments, a group of formula (iv) is of formula

wherein q is an integer between 1 and 50, inclusive, and each of R⁶ andR⁷ is independently as defined above and described herein.

As generally defined above, p is an integer of between 1 and 9,inclusive. In certain embodiments, p is 1. In certain embodiments, p is2. In certain embodiments, p is 3. In certain embodiments, p is 4. Incertain embodiments, p is 5. In certain embodiments, p is 6. In certainembodiments, p is 7. In certain embodiments, p is 8. In certainembodiments, p is 9.

In some embodiments, p is 1. In some embodiments, a compound of formulaI is a compound of formula (I-a):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 2. In some embodiments, a compound of formulaI is a compound of formula (I-p2):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 3. In some embodiments, a compound of formulaI is a compound of formula (I-p3):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 4. In some embodiments, a compound of formulaI is a compound of formula (I-p4):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 5. In some embodiments, a compound of formulaI is a compound of formula (I-p5):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 6. In some embodiments, a compound of formulaI is a compound of formula (I-p6):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 7. In some embodiments, a compound of formulaI is a compound of formula (I-p7):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 8. In some embodiments, a compound of formulaI is a compound of formula (I-p8):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, p is 9. In some embodiments, a compound of formulaI is a compound of formula (I-p9):

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

As generally defined above, each instance of Q is independently O, S, orNR^(Q), wherein R^(Q) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii), or(iii).

In certain embodiments, at least one instance of Q is O. In certainembodiments, each instance of Q is O. In certain embodiments, at leastone instance of Q is S. In certain embodiments, each instance of Q is S.In certain embodiments, at least one instance of Q is NR^(Q), whereinR^(Q) is as defined above and described herein. In certain embodiments,each instance of Q is NR^(Q), wherein each R^(Q) is independently asdefined above and described herein. In certain embodiments, eachinstance of R^(Q) is independently hydrogen or a group of the formula(i), (ii), or (iii).

As generally defined above, R^(Q) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of the formula (i), (ii) or (iii).

In some embodiments, R^(Q) is hydrogen. In some embodiments, R^(Q) isoptionally substituted alkyl. In some embodiments, R^(Q) is optionallysubstituted alkenyl. In some embodiments, R^(Q) is optionallysubstituted alkynyl. In some embodiments, R^(Q) is carbocyclyl. In someembodiments, R^(Q) is optionally substituted heterocyclyl. In someembodiments, R^(Q) is optionally substituted aryl. In some embodiments,R^(Q) is optionally substituted heteroaryl. In some embodiments, R^(Q)is a nitrogen protecting group. In some embodiments, R^(Q) is a group offormula (i), (ii) or (iii). In some embodiments, R^(Q) is a group offormula (i). In some embodiments, R^(Q) is a group of formula (ii). Insome embodiments, R^(Q) is a group of formula (iii).

As generally defined above, each instance of R¹ is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, halogen, —OR^(A1), —N(R^(A1))₂, or—SR^(A1), or a group of formula (iv), wherein each of R^(A1) and formula(iv) is independently as defined above and described herein.

In some embodiments, R¹ is hydrogen.

In certain embodiments, R¹ is optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, or optionally substituted heteroaryl. In certainembodiments, at least one instance of R¹ is optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl.

In certain embodiments, R¹ is optionally substituted alkyl; e.g.,optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkyl,optionally substituted C₃₋₆alkyl, optionally substituted C₄₋₆alkyl,optionally substituted C₄₋₅alkyl, or optionally substituted C₃₋₄alkyl.In certain embodiments, at least one instance of R¹ is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, R¹ is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl,optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl,or optionally substituted C₃₋₄alkenyl. In certain embodiments, at leastone instance of R¹ is optionally substituted alkenyl, e.g., optionallysubstituted C₂₋₆ alkenyl, optionally substituted C₃₋₆alkenyl, optionallysubstituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl, oroptionally substituted C₃₋₄alkenyl.

In certain embodiments, R¹ is optionally substituted alkynyl, e.g.,optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl,optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl,or optionally substituted C₃₋₄alkynyl. In certain embodiments, at leastone instance of R¹ is optionally substituted alkynyl, e.g., optionallysubstituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl, optionallysubstituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl, oroptionally substituted C₃₋₄alkynyl.

In certain embodiments, R¹ is optionally substituted carbocyclyl, e.g.,optionally substituted C₃₋₁₀ carbocyclyl, optionally substituted C₅₋₈carbocyclyl, optionally substituted C₅₋₆ carbocyclyl, optionallysubstituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl. Incertain embodiments, at least one instance of R¹ is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀ carbocyclyl,optionally substituted C₅₋₈ carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In some embodiments, R¹ is optionally substituted heterocyclyl, e.g.,optionally substituted 3-14 membered heterocyclyl, optionallysubstituted 3-10 membered heterocyclyl, optionally substituted 5-8membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl. In certain embodiments, at leastone instance of R¹ is optionally substituted heterocyclyl, e.g.,optionally substituted 3-14 membered heterocyclyl, optionallysubstituted 3-10 membered heterocyclyl, optionally substituted 5-8membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl.

In some embodiments, R¹ is optionally substituted aryl. In someembodiments, R¹ is optionally substituted phenyl. In some embodiments,R¹ is phenyl. In some embodiments, R¹ is substituted phenyl. In certainembodiments, at least one instance of R¹ is optionally substituted aryl,e.g., optionally substituted phenyl.

In some embodiments, R¹ is optionally substituted heteroaryl, e.g.,optionally substituted 5-14 membered heteroaryl, optionally substituted5-10 membered heteroaryl, optionally substituted 5-6 memberedheteroaryl, optionally substituted 5 membered heteroaryl, or optionallysubstituted 6 membered heteroaryl. In certain embodiments, at least oneinstance of R¹ is optionally substituted heteroaryl, e.g., optionallysubstituted 5-14 membered heteroaryl, optionally substituted 5-10membered heteroaryl, optionally substituted 5-6 membered heteroaryl,optionally substituted 5 membered heteroaryl, or optionally substituted6 membered heteroaryl.

In some embodiments, R¹ is halogen. In some embodiments, R¹ is —F. Insome embodiments, R¹ is —Cl. In some embodiments, R¹ is —Br. In someembodiments, R¹ is —I.

In some embodiments, R¹ is —OR^(A1), wherein R^(A1) is as defined aboveand described herein. In some embodiments, R¹ is —N(R^(A1))₂, whereineach R^(A1) is independently as defined above and described herein. Insome embodiments, R¹ is —SR^(A1), wherein R^(A1) is as defined above anddescribed herein.

In some embodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, or heteroaryl group may be substituted. In someembodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, or heteroaryl group may be substituted with an optionallysubstituted amino group. In some embodiments, an R¹ alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl group may besubstituted with an optionally substituted hydroxyl group. In someembodiments, an R¹ alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl,aryl, or heteroaryl group may be substituted with an optionallysubstituted thiol group. In any of the above embodiments, an R¹ alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl groupmay be substituted, for example, with an optionally substituted aminogroup (e.g., —NR⁶R⁷), an optionally substituted hydroxyl group (e.g.,—OR⁶), an optionally substituted thiol group (e.g., —SR⁶), or with agroup of formula (i), (ii), or (iii), wherein each instance of R⁶ and R⁷is independently hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, a nitrogenprotecting group when attached to a nitrogen atom, an oxygen protectinggroup when attached to an oxygen atom, and a sulfur protecting groupwhen attached to a sulfur atom, or a group of formula (i), (ii), or(iii).

In some embodiments, R¹ is an optionally substituted natural amino acidside chain. In some embodiments, R¹ is a natural amino acid side chain.In some embodiments, R¹ is an optionally substituted unnatural aminoacid side chain. In some embodiments, R¹ is an unnatural amino acid sidechain.

In certain embodiments, each instance of R¹ is the same. In certainembodiments, at least one R¹ group is different. In certain embodiments,each R¹ group is different.

In certain embodiments, R¹ is an alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, or heteroaryl group substituted with an amino groupof the formula —NR⁶R⁷.

In certain embodiments, R¹ is a group of formula (iv):

wherein:L is an optionally substituted alkylene, optionally substitutedalkenylene, optionally substituted alkynylene, optionally substitutedheteroalkylene, optionally substituted heteroalkenylene, optionallysubstituted heteroalkynylene, optionally substituted carbocyclylene,optionally substituted heterocyclylene, optionally substituted arylene,or optionally substituted heteroarylene, or combination thereof, andeach of R⁶ and R⁷ is independently hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anitrogen protecting group, or a group of formula (i), (ii) or (iii):

wherein each of R′, Y, R^(P), R^(L) and X is independently as definedabove and described herein.

In some embodiments, at least one instance of R¹ is an alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, or heteroaryl groupsubstituted with an amino group of the formula —NR⁶R⁷. In someembodiments, at least one instance of R¹ is a group of formula (iv). Insome embodiments, at least one instance of R¹ is a group of formula(iv), wherein at least one instance of R⁶ and R⁷ is a group of theformula (i), (ii) or (iii). In some embodiments, at least one instanceof R¹ is a group of formula (iv), wherein each instance of R⁶ and R⁷ isa group of the formula (i), (ii) or (iii). In some embodiments, at leastone instance of R¹ is a group of formula (iv), wherein each instance ofR⁶ and R⁷ is a group of the formula (i). In some embodiments, at leastone instance of R¹ is a group of formula (iv), wherein each instance ofR⁶ and R⁷ is a group of the formula (ii). In some embodiments, at leastone instance of R¹ is a group of formula (iv), wherein each instance ofR⁶ and R⁷ is a group of the formula (iii).

In some embodiments, each instance of R¹ is a group of formula (iv). Insome embodiments, each instance of R¹ is a group of formula (iv),wherein each instance of R⁶ and R⁷ is a group of the formula (i), (ii)or (iii). In some embodiments, each instance of R¹ is a group of formula(iv), wherein each instance of R⁶ and R⁷ is a group of the formula (i),(ii) or (iii). In some embodiments, each instance of R¹ is a group offormula (iv), wherein each instance of R⁶ and R⁷ is a group of theformula (i). In some embodiments, each instance of R¹ is a group offormula (iv), wherein each instance of R⁶ and R⁷ is a group of theformula (ii). In some embodiments, each instance of R¹ is a group offormula (iv), wherein each instance of R⁶ and R⁷ is a group of theformula (iii).

In certain embodiments, at least two instances of R¹ is a group offormula (iv). In certain embodiments, at least three instances of R¹ isa group of formula (iv). In certain embodiments, at least four instancesof R¹ is a group of formula (iv). In certain embodiments, at least fiveinstances of R¹ is a group of formula (iv). In certain embodiments, atleast six instances of R¹ is a group of formula (iv). In certainembodiments, at least seven instances of R¹ is a group of formula (iv).In certain embodiments, at least eight instances of R¹ is a group offormula (iv). In certain embodiments, at least nine instances of R¹ is agroup of formula (iv). In certain embodiments, each instance of R¹ is agroup of formula (iv).

In certain embodiments, L is an optionally substituted alkylene; e.g.,optionally substituted C₁₋₅₀alkylene, optionally substitutedC₁₋₄₀alkylene, optionally substituted C₁₋₃₀alkylene, optionallysubstituted C₁₋₂₀alkylene, optionally substituted C₄₋₂₀alkylene,optionally substituted C₆₋₂₀alkylene, optionally substitutedC₈₋₂₀alkylene, optionally substituted C₁₀₋₂₀alkylene, optionallysubstituted C₁₋₆alkylene, optionally substituted C₂₋₆alkylene,optionally substituted C₃₋₆alkylene, optionally substitutedC₄₋₆alkylene, optionally substituted C₄₋₅alkylene, or optionallysubstituted C₃₋₄alkylene. In some embodiments, L is optionallysubstituted C₁ alkylene. In some embodiments, L is optionallysubstituted C₂ alkylene. In some embodiments, L is optionallysubstituted C₃ alkylene. In some embodiments, L is optionallysubstituted C₄ alkylene. In some embodiments, L is optionallysubstituted C₅ alkylene. In some embodiments, L is optionallysubstituted C₆ alkylene. In some embodiments, L is optionallysubstituted C₇ alkylene. In some embodiments, L is optionallysubstituted C₈ alkylene. In some embodiments, L is optionallysubstituted C₉ alkylene. In some embodiments, L is optionallysubstituted C₁₀ alkylene. In some embodiments, L is —CH₂—. In someembodiments, L is —(CH₂)₂—. In some embodiments, L is —(CH₂)₃—. In someembodiments, L is —(CH₂)₄—. In some embodiments, L is —(CH₂)₅—. In someembodiments, L is —(CH₂)₆—. In some embodiments, L is —(CH₂)₇—. In someembodiments, L is —(CH₂)₈—. In some embodiments, L is —(CH₂)₉—. In someembodiments, L is —(CH₂)₁₀—.

In certain embodiments, L is an optionally substituted alkenylene, e.g.,optionally substituted C₂₋₅₀alkenylene, optionally substitutedC₂₋₄₀alkenylene, optionally substituted C₂₋₃₀alkenylene, optionallysubstituted C₂₋₂₀alkenylene, optionally substituted C₄₋₂₀alkenylene,optionally substituted C₆₋₂₀alkenylene, optionally substitutedC₈₋₂₀alkenylene, optionally substituted C₁₀₋₂₀alkenylene, optionallysubstituted C₂₋₆alkenylene, optionally substituted C₃₋₆ alkenylene,optionally substituted C₄₋₆alkenylene, optionally substitutedC₄₋₅alkenylene, or optionally substituted C₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted alkynylene, e.g.,optionally substituted C₂₋₅₀alkynylene, optionally substitutedC₂₋₄₀alkynylene, optionally substituted C₂₋₃₀alkynylene, optionallysubstituted C₂₋₂₀alkynylene, optionally substituted C₄₋₂₀alkynylene,optionally substituted C₆₋₂₀alkynylene, optionally substitutedC₈₋₂₀alkynylene, optionally substituted C₁₀₋₂₀alkynylene, optionallysubstituted C₂₋₆alkynylene, optionally substituted C₃₋₆alkynylene,optionally substituted C₄₋₆alkynylene, optionally substitutedC₄₋₅alkynylene, or optionally substituted C₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted heteroalkylene;e.g., optionally substituted heteroC₁₋₅₀alkylene, optionally substitutedheteroC₁₋₄₀alkylene, optionally substituted heteroC₁₋₃₀alkylene,optionally substituted heteroC₁₋₂₀alkylene, optionally substitutedheteroC₄₋₂₀alkylene, optionally substituted heteroC₆₋₂₀alkylene,optionally substituted heteroC₈₋₂₀alkylene, optionally substitutedheteroC₁₀₋₂₀alkylene, optionally substituted heteroC₁₋₆alkylene,optionally substituted heteroC₂₋₆alkylene, optionally substitutedheteroC₃₋₆alkylene, optionally substituted heteroC₄₋₆alkylene,optionally substituted heteroC₄₋₅alkylene, or optionally substitutedheteroC₃₋₄alkylene. In some embodiments, L is optionally substitutedheteroC₂alkylene. In some embodiments, L is optionally substitutedheteroC₃alkylene. In some embodiments, L is optionally substitutedheteroC₄alkylene. In some embodiments, L is optionally substitutedheteroC₅alkylene. In some embodiments, L is optionally substitutedheteroC₆alkylene. In some embodiments, L is optionally substitutedheteroC₇alkylene. In some embodiments, L is optionally substitutedheteroC₈alkylene. In some embodiments, L is optionally substitutedheteroC₉alkylene. In some embodiments, L is optionally substitutedheteroC₁₀alkylene.

In certain embodiments, L is an optionally substituted heteroalkenylene,e.g., optionally substituted heteroC₂₋₅₀alkenylene, optionallysubstituted heteroC₂₋₄₀alkenylene, optionally substitutedheteroC₂₋₃₀alkenylene, optionally substituted heteroC₂₋₂₀alkenylene,optionally substituted heteroC₄₋₂₀alkenylene, optionally substitutedheteroC₆₋₂₀alkenylene, optionally substituted heteroC₈₋₂₀alkenylene,optionally substituted heteroC₁₀₋₂₀alkenylene, optionally substitutedheteroC₂₋₆alkenylene, optionally substituted heteroC₃₋₆alkenylene,optionally substituted heteroC₄₋₆alkenylene, optionally substitutedheteroC₄₋₅alkenylene, or optionally substituted heteroC₃₋₄alkenylene.

In certain embodiments, L is an optionally substituted heteroalkynylene,e.g., optionally substituted heteroC₂₋₅₀alkynylene, optionallysubstituted heteroC₂₋₄₀alkynylene, optionally substitutedheteroC₂₋₃₀alkynylene, optionally substituted heteroC₂₋₂₀alkynylene,optionally substituted heteroC₄₋₂₀alkynylene, optionally substitutedheteroC₆₋₂₀alkynylene, optionally substituted heteroC₈₋₂₀alkynylene,optionally substituted heteroC₁₀₋₂₀alkynylene, optionally substitutedheteroC₂₋₆alkynylene, optionally substituted heteroC₃₋₆alkynylene,optionally substituted heteroC₄₋₆alkynylene, optionally substitutedheteroC₄₋₅alkynylene, or optionally substituted heteroC₃₋₄alkynylene.

In certain embodiments, L is an optionally substituted carbocyclylene,e.g., optionally substituted C₃₋₁₀carbocyclylene, optionally substitutedC₅₋₈carbocyclylene, optionally substituted C₅₋₆carbocyclylene,optionally substituted C₅carbocyclylene, or optionally substitutedC₆carbocyclylene.

In certain embodiments, L is an optionally substituted heterocyclylene,e.g., optionally substituted 3-14 membered heterocyclylene, optionallysubstituted 3-10 membered heterocyclylene, optionally substituted 5-8membered heterocyclylene, optionally substituted 5-6 memberedheterocyclylene, optionally substituted 5-membered heterocyclylene, oroptionally substituted 6-membered heterocyclylene.

In certain embodiments, L is an optionally substituted arylene, e.g.,optionally substituted phenylene. In some embodiments, L is optionallysubstituted phenylene. In some embodiments, L is substituted phenylene.In some embodiments, L is unsubstituted phenylene.

In certain embodiments, L is an optionally substituted heteroarylene,e.g., optionally substituted 5-14 membered heteroarylene, optionallysubstituted 5-10 membered heteroarylene, optionally substituted 5-6membered heteroarylene, optionally substituted 5-membered heteroarylene,or optionally substituted 6-membered heteroarylene.

In certain embodiments, wherein L is an optionally substituted alkylenegroup, the group of formula (iv) is a group of the formula

wherein q is an integer between 1 and 50, inclusive, and each of R⁶ andR⁷ is independently as defined above and described herein.

In certain embodiments, q is an integer between 1 and 40, inclusive. Incertain embodiments, q is an integer between 1 and 30, inclusive. Incertain embodiments, q is an integer between 1 and 20, inclusive. Incertain embodiments, q is an integer between 1 and 10, inclusive. Incertain embodiments, q is an integer between 4 and 20, inclusive. Incertain embodiments, q is an integer between 6 and 20, inclusive. Incertain embodiments, q is an integer between 2 and 10, inclusive. Incertain embodiments, q is an integer between 2 and 9, inclusive. Incertain embodiments, q is an integer between 2 and 8, inclusive. Incertain embodiments, q is an integer between 2 and 7, inclusive. Incertain embodiments, q is an integer between 2 and 6, inclusive. Incertain embodiments, q is an integer between 2 and 5, inclusive. Incertain embodiments, q is an integer between 2 and 4, inclusive. Incertain embodiments, q is an integer between 3 and 10, inclusive. Incertain embodiments, q is an integer between 3 and 8, inclusive. Incertain embodiments, q is an integer between 3 and 7, inclusive. Incertain embodiments, q is an integer between 3 and 6, inclusive. Incertain embodiments, q is an integer between 3 and 5, inclusive. Incertain embodiments, q is 3 or 4. In certain embodiments, q is aninteger between 3 and 9, inclusive. In certain embodiments, q is aninteger between 8 and 20, inclusive. In certain embodiments, q is 1. Incertain embodiments, q is 2. In certain embodiments, q is 3. In certainembodiments, q is 4. In certain embodiments, q is 5. In certainembodiments, q is 6. In certain embodiments, q is 7. In certainembodiments, q is 8. In certain embodiments, q is 9. In certainembodiments, q is 10.

As generally defined above, each R⁶ is independently selected from thegroup consisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, a nitrogenprotecting group, or a group of formula (i), (ii) or (iii).

In some embodiments, R⁶ is hydrogen.

In some embodiments, R⁶ is optionally substituted alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₂ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₁ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₀ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₉ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₈ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₇ alkyl. In someembodiments, R⁶ is optionally substituted C₂₋₆ alkyl.

In some embodiments, R⁶ is optionally substituted C₄₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₂ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₁ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₀ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₉ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₈ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₇ alkyl. In someembodiments, R⁶ is optionally substituted C₄₋₆ alkyl.

In some embodiments, R⁶ is optionally substituted C₆₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₂ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₁ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₀ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₉ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₈ alkyl. In someembodiments, R⁶ is optionally substituted C₆₋₇ alkyl.

In some embodiments, R⁶ is optionally substituted C₈₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₂ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₁ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₀ alkyl. In someembodiments, R⁶ is optionally substituted C₈₋₉ alkyl.

In some embodiments, R⁶ is optionally substituted C₉₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₂ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₁ alkyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₀ alkyl.

In some embodiments, R⁶ is optionally substituted C₁₀₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₂ alkyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₁ alkyl.

In some embodiments, R⁶ is optionally substituted C₁₁₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₂ alkyl.

In some embodiments, R⁶ is optionally substituted C₁₂₋₅₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₄₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₃₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₂₀ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₃ alkyl.

In some embodiments, R⁶ is optionally substituted C₆ alkyl. In someembodiments, R⁶ is optionally substituted C₇ alkyl. In some embodiments,R⁶ is optionally substituted C₈ alkyl. In some embodiments, R⁶ isoptionally substituted C₉ alkyl. In some embodiments, R⁶ is optionallysubstituted C₁₀ alkyl. In some embodiments, R⁶ is optionally substitutedC₁₁ alkyl. In some embodiments, R⁶ is optionally substituted C₁₂ alkyl.In some embodiments, R⁶ is optionally substituted C₁₃ alkyl. In someembodiments, R⁶ is optionally substituted C₁₄ alkyl. In someembodiments, R⁶ is optionally substituted C₁₅ alkyl. In someembodiments, R⁶ is optionally substituted C₁₆ alkyl. In someembodiments, R⁶ is optionally substituted C₁₇ alkyl. In someembodiments, R⁶ is optionally substituted C₁₈ alkyl. In someembodiments, R⁶ is optionally substituted C₁₉ alkyl. In someembodiments, R⁶ is optionally substituted C₂₀ alkyl.

In some embodiments, for example, in any of the above embodiments, R⁶ isa substituted alkyl group. In some embodiments, R⁶ is an unsubstitutedalkyl group. In some embodiments, R⁶ is an optionally substitutedstraight-chain alkyl group. In some embodiments, R⁶ is a substitutedstraight-chain alkyl group. In some embodiments, R⁶ is an unsubstitutedstraight-chain alkyl group. In some embodiments, R⁶ is an optionallysubstituted branched alkyl group. In some embodiments, R⁶ is asubstituted branched alkyl group. In some embodiments, R⁶ is anunsubstituted branched alkyl group.

In some embodiments, R⁶ is optionally substituted alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₅₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₁ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₋₆ alkenyl.

In some embodiments, R⁶ is optionally substituted C₄₋₅₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₁ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₄₋₆ alkenyl.

In some embodiments, R⁶ is optionally substituted C₆₋₅₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₁ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₆₋₇ alkenyl.

In some embodiments, R⁶ is optionally substituted C₈₋₅₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₁ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₈₋₉ alkenyl.

In some embodiments, R⁶ is optionally substituted C₉₋₅₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₁ alkenyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₀ alkenyl.

In some embodiments, R⁶ is optionally substituted C₁₀₋₅₀ alkenyl. Insome embodiments, R⁶ is optionally substituted C₁₀₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₁ alkenyl.

In some embodiments, R⁶ is optionally substituted C₁₁₋₅₀ alkenyl. Insome embodiments, R⁶ is optionally substituted C₁₁₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₂ alkenyl.

In some embodiments, R⁶ is optionally substituted C₁₂₋₅₀ alkenyl. Insome embodiments, R⁶ is optionally substituted C₁₂₋₄₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₃₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₂₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₃ alkenyl.

In some embodiments, R⁶ is optionally substituted C₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₀ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₁ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₂ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₃ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₄ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₅ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₆ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₇ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₈ alkenyl. In someembodiments, R⁶ is optionally substituted C₁₉ alkenyl. In someembodiments, R⁶ is optionally substituted C₂₀ alkenyl.

In some embodiments, for example, in any of the above embodiments, R⁶ isa substituted alkenyl group. In some embodiments, R⁶ is an unsubstitutedalkenyl group. In some embodiments, R⁶ is an optionally substitutedstraight-chain alkenyl group. In some embodiments, R⁶ is a substitutedstraight-chain alkenyl group. In some embodiments, R⁶ is anunsubstituted straight-chain alkenyl group. In some embodiments, R⁶ isan optionally substituted branched alkenyl group. In some embodiments,R⁶ is a substituted branched alkenyl group. In some embodiments, R⁶ isan unsubstituted branched alkenyl group.

In some embodiments, R⁶ is optionally substituted alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₅₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₁ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₁₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₋₆ alkynyl.

In some embodiments, R⁶ is optionally substituted C₄₋₅₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₁ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₁₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₄₋₆ alkynyl.

In some embodiments, R⁶ is optionally substituted C₆₋₅₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₁ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₁₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₆₋₇ alkynyl.

In some embodiments, R⁶ is optionally substituted C₈₋₅₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₁ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₁₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₈₋₉ alkynyl.

In some embodiments, R⁶ is optionally substituted C₉₋₅₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₁ alkynyl. In someembodiments, R⁶ is optionally substituted C₉₋₁₀ alkynyl.

In some embodiments, R⁶ is optionally substituted C₁₀₋₅₀ alkynyl. Insome embodiments, R⁶ is optionally substituted C₁₀₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀₋₁₁ alkynyl.

In some embodiments, R⁶ is optionally substituted C₁₁₋₅₀ alkynyl. Insome embodiments, R⁶ is optionally substituted C₁₁₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁₋₁₂ alkynyl.

In some embodiments, R⁶ is optionally substituted C₁₂₋₅₀ alkynyl. Insome embodiments, R⁶ is optionally substituted C₁₂₋₄₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₃₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₂₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂₋₁₃ alkynyl.

In some embodiments, R⁶ is optionally substituted C₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₀ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₁ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₂ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₃ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₄ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₅ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₆ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₇ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₈ alkynyl. In someembodiments, R⁶ is optionally substituted C₁₉ alkynyl. In someembodiments, R⁶ is optionally substituted C₂₀ alkynyl.

In some embodiments, for example, in any of the above embodiments, R⁶ isa substituted alkynyl group. In some embodiments, R⁶ is an unsubstitutedalknyl group. In some embodiments, R⁶ is an optionally substitutedstraight-chain alkynyl group. In some embodiments, R⁶ is a substitutedstraight-chain alkynyl group. In some embodiments, R⁶ is anunsubstituted straight-chain alkynyl group. In some embodiments, R⁶ isan optionally substituted branched alkynyl group. In some embodiments,R⁶ is a substituted branched alkynyl group. In some embodiments, R⁶ isan unsubstituted branched alkynyl group.

In some embodiments, R⁶ is optionally substituted carbocyclyl. In someembodiments, R⁶ is optionally substituted heterocyclyl. In someembodiments, R⁶ is optionally substituted aryl. In some embodiments, R⁶is optionally substituted heteroaryl. In some embodiments, R⁶ is anitrogen protecting group.

In some embodiments, R⁶ is a group of formula (i). In some embodiments,R⁶ is a group of formula (i-a). In some embodiments, R⁶ is a group offormula

In some embodiments, R⁶ is a group of formula (i-b). In someembodiments, R⁶ is a group of formula (ii). In some embodiments, R⁶ is agroup of formula (iii).

In some embodiments, R⁶ is substituted with one or more hydroxyl groups.In some embodiments, R⁶ is substituted with one hydroxyl group. In someembodiments, R⁶ is substituted with one 2-hydroxyl group (C1 is thecarbon atom directly bonded to the nitrogen atom depicted in formula(iv)).

As generally defined above, each R⁷ is independently selected from thegroup consisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted carbocyclyl, optionally substituted heterocyclyl, optionallysubstituted aryl, optionally substituted heteroaryl, a nitrogenprotecting group, or a group of formula (i), (ii) or (iii).

In some embodiments, R⁷ is hydrogen.

In some embodiments, R⁷ is optionally substituted alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₂ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₁ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₀ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₉ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₈ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₇ alkyl. In someembodiments, R⁷ is optionally substituted C₂₋₆ alkyl.

In some embodiments, R⁷ is optionally substituted C₄₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₂ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₁ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₀ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₉ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₈ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₇ alkyl. In someembodiments, R⁷ is optionally substituted C₄₋₆ alkyl.

In some embodiments, R⁷ is optionally substituted C₆₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₂ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₁ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₀ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₉ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₈ alkyl. In someembodiments, R⁷ is optionally substituted C₆₋₇ alkyl.

In some embodiments, R⁷ is optionally substituted C₈₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₂ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₁ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₀ alkyl. In someembodiments, R⁷ is optionally substituted C₈₋₉ alkyl.

In some embodiments, R⁷ is optionally substituted C₉₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₂ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₁ alkyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₀ alkyl.

In some embodiments, R⁷ is optionally substituted C₁₀₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₂ alkyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₁ alkyl.

In some embodiments, R⁷ is optionally substituted C₁₁₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₂ alkyl.

In some embodiments, R⁷ is optionally substituted C₁₂₋₅₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₄₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₃₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₂₀ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₃ alkyl.

In some embodiments, R⁷ is optionally substituted C₆ alkyl. In someembodiments, R⁷ is optionally substituted C₇ alkyl. In some embodiments,R⁷ is optionally substituted C₈ alkyl. In some embodiments, R⁷ isoptionally substituted C₉ alkyl. In some embodiments, R⁷ is optionallysubstituted C₁₀ alkyl. In some embodiments, R⁷ is optionally substitutedC₁₁ alkyl. In some embodiments, R⁷ is optionally substituted C₁₂ alkyl.In some embodiments, R⁷ is optionally substituted C₁₃ alkyl. In someembodiments, R⁷ is optionally substituted C₁₄ alkyl. In someembodiments, R⁷ is optionally substituted C₁₅ alkyl. In someembodiments, R⁷ is optionally substituted C₁₆ alkyl. In someembodiments, R⁷ is optionally substituted C₁₇ alkyl. In someembodiments, R⁷ is optionally substituted C₁₈ alkyl. In someembodiments, R⁷ is optionally substituted C₁₉ alkyl. In someembodiments, R⁷ is optionally substituted C₂₀ alkyl.

In some embodiments, for example, in any of the above embodiments, R⁷ isa substituted alkyl group. In some embodiments, R⁷ is an unsubstitutedalkyl group. In some embodiments, R⁷ is an optionally substitutedstraight-chain alkyl group. In some embodiments, R⁷ is a substitutedstraight-chain alkyl group. In some embodiments, R⁷ is an unsubstitutedstraight-chain alkyl group. In some embodiments, R⁷ is an optionallysubstituted branched alkyl group. In some embodiments, R⁷ is asubstituted branched alkyl group. In some embodiments, R⁷ is anunsubstituted branched alkyl group.

In some embodiments, R⁷ is optionally substituted alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₅₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₁ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₋₆ alkenyl.

In some embodiments, R⁷ is optionally substituted C₄₋₅₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₁ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₄₋₆ alkenyl.

In some embodiments, R⁷ is optionally substituted C₆₋₅₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₁ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₆₋₇ alkenyl.

In some embodiments, R⁷ is optionally substituted C₈₋₅₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₁ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₈₋₉ alkenyl.

In some embodiments, R⁷ is optionally substituted C₉₋₅₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₁ alkenyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₀ alkenyl.

In some embodiments, R⁷ is optionally substituted C₁₀₋₅₀ alkenyl. Insome embodiments, R⁷ is optionally substituted C₁₀₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₁ alkenyl.

In some embodiments, R⁷ is optionally substituted C₁₁₋₅₀ alkenyl. Insome embodiments, R⁷ is optionally substituted C₁₁₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₂ alkenyl.

In some embodiments, R⁷ is optionally substituted C₁₂₋₅₀ alkenyl. Insome embodiments, R⁷ is optionally substituted C₁₂₋₄₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₃₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₂₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₃ alkenyl.

In some embodiments, R⁷ is optionally substituted C₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₀ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₁ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₂ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₃ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₄ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₅ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₆ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₇ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₈ alkenyl. In someembodiments, R⁷ is optionally substituted C₁₉ alkenyl. In someembodiments, R⁷ is optionally substituted C₂₀ alkenyl.

In some embodiments, for example, in any of the above embodiments, R⁷ isa substituted alkenyl group. In some embodiments, R⁷ is an unsubstitutedalkenyl group. In some embodiments, R⁷ is an optionally substitutedstraight-chain alkenyl group. In some embodiments, R⁷ is a substitutedstraight-chain alkenyl group. In some embodiments, R⁷ is anunsubstituted straight-chain alkenyl group. In some embodiments, R⁷ isan optionally substituted branched alkenyl group. In some embodiments,R⁷ is a substituted branched alkenyl group. In some embodiments, R⁷ isan unsubstituted branched alkenyl group.

In some embodiments, R⁷ is optionally substituted alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₅₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₁ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₁₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₋₆ alkynyl.

In some embodiments, R⁷ is optionally substituted C₄₋₅₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₁ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₁₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₄₋₆ alkynyl.

In some embodiments, R⁷ is optionally substituted C₆₋₅₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₁ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₁₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₆₋₇ alkynyl.

In some embodiments, R⁷ is optionally substituted C₈₋₅₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₁ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₁₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₈₋₉ alkynyl.

In some embodiments, R⁷ is optionally substituted C₉₋₅₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₁ alkynyl. In someembodiments, R⁷ is optionally substituted C₉₋₁₀ alkynyl.

In some embodiments, R⁷ is optionally substituted C₁₀₋₅₀ alkynyl. Insome embodiments, R⁷ is optionally substituted C₁₀₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀₋₁₁ alkynyl.

In some embodiments, R⁷ is optionally substituted C₁₁₋₅₀ alkynyl. Insome embodiments, R⁷ is optionally substituted C₁₁₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁₋₁₂ alkynyl.

In some embodiments, R⁷ is optionally substituted C₁₂₋₅₀ alkynyl. Insome embodiments, R⁷ is optionally substituted C₁₂₋₄₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₃₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₂₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂₋₁₃ alkynyl.

In some embodiments, R⁷ is optionally substituted C₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₀ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₁ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₂ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₃ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₄ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₅ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₆ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₇ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₈ alkynyl. In someembodiments, R⁷ is optionally substituted C₁₉ alkynyl. In someembodiments, R⁷ is optionally substituted C₂₀ alkynyl.

In some embodiments, for example, in any of the above embodiments, R⁷ isa substituted alkynyl group. In some embodiments, R⁷ is an unsubstitutedalkynyl group. In some embodiments, R⁷ is an optionally substitutedstraight-chain alkynyl group. In some embodiments, R⁷ is a substitutedstraight-chain alkynyl group. In some embodiments, R⁷ is anunsubstituted straight-chain alkynyl group. In some embodiments, R⁷ isan optionally substituted branched alkynyl group. In some embodiments,R⁷ is a substituted branched alkynyl group. In some embodiments, R⁷ isan unsubstituted branched alkynyl group.

In some embodiments, R⁷ is optionally substituted carbocyclyl. In someembodiments, R⁷ is optionally substituted heterocyclyl. In someembodiments, R⁷ is optionally substituted aryl. In some embodiments, R⁷is optionally substituted heteroaryl. In some embodiments, R⁷ is anitrogen protecting group.

In some embodiments, R⁷ is a group of formula (i). In some embodiments,R⁷ is a group of formula (i-a). In some embodiments, R⁷ is a group offormula

In some embodiments, R⁷ is a group of formula (i-b). In someembodiments, R⁷ is a group of formula (ii). In some embodiments, R⁷ is agroup of formula (iii).

In some embodiments, at least one instance of R⁶ and R⁷ is a group ofthe formula (i), (ii) or (iii). In some embodiments, each instance of R⁶and R⁷ is independently a group of the formula (i), (ii) or (iii). Insome embodiments, each instance of R⁶ and R⁷ is independently a group ofthe formula (i). In some embodiments, each instance of R⁶ and R⁷ isindependently a group of the formula (i-a). In some embodiments, eachinstance of R⁶ and R⁷ is independently a group of the formula (i-b). Insome embodiments, each instance of R⁶ and R⁷ is independently a group ofthe formula (ii). In some embodiments, each instance of R⁶ and R⁷ isindependently a group of the formula (iii).

In some embodiments, R⁶ and R⁷ are the same. In some embodiments, R⁶ andR⁷ are different.

In certain embodiments, both R⁶ and R⁷ are hydrogen. In certainembodiments, R⁶ is hydrogen and R⁷ is a group of the formula (i), (ii),or (iii). In certain embodiments, R⁶ is hydrogen and R⁷ is a group ofthe formula (i). In certain embodiments, R⁶ is hydrogen and R⁷ is agroup of the formula (ii). In certain embodiments, R⁶ is hydrogen and R⁷is a group of the formula (iii). In certain embodiments, each of R⁶ andR⁷ is independently a group of the formula (i), (ii), or (iii). Incertain embodiments, each of R⁶ and R⁷ is independently a group of theformula (i). In certain embodiments, each of R⁶ and R⁷ is independentlya group of the formula (ii). In certain embodiments, each of R⁶ and R⁷is independently a group of the formula (iii). In certain embodiments,R⁶ and R⁷ are the same group, which is selected from formulas (i), (ii),and (iii). In some embodiments, R⁶ and R⁷ are the same group of formula(i). In some embodiments, R⁶ and R⁷ are the same group of formula (i-a).In some embodiments, R⁶ and R⁷ are the same group of formula (i-a1). Insome embodiments, R⁶ and R⁷ are the same group of formula (i-b).

In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is as defined above and described herein. In someembodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₋₅₀alkyl, optionallysubstituted C₂₋₅₀alkenyl, optionally substituted C₂₋₅₀alkynyl,optionally substituted heteroC₁₋₅₀alkyl, optionally substitutedheteroC₂₋₅₀alkenyl, or optionally substituted heteroC₂₋₅₀alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₅₀alkyl, optionallysubstituted C₅₋₅₀alkenyl, optionally substituted C₅₋₅₀alkynyl,optionally substituted heteroC₅₋₅₀alkyl, optionally substitutedheteroC₅₋₅₀alkenyl, or optionally substituted heteroC₅₋₅₀alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₄₀alkyl, optionallysubstituted C₅₋₄₀alkenyl, optionally substituted C₅₋₄₀alkynyl,optionally substituted heteroC₅₋₄₀alkyl, optionally substitutedheteroC₅₋₄₀alkenyl, or optionally substituted heteroC₅₋₄₀alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₃₀alkyl, optionallysubstituted C₅₋₃₀alkenyl, optionally substituted C₅₋₃₀alkynyl,optionally substituted heteroC₅₋₃₀alkyl, optionally substitutedheteroC₅₋₃₀alkenyl, or optionally substituted heteroC₅₋₃₀alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₅alkyl, optionallysubstituted C₅₋₂₅alkenyl, optionally substituted C₅₋₂₅alkynyl,optionally substituted heteroC₅₋₂₅alkyl, optionally substitutedheteroC₅₋₂₅alkenyl, or optionally substituted heteroC₅₋₂₅alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₀alkyl, optionallysubstituted C₅₋₂₀alkenyl, optionally substituted C₅₋₂₀alkynyl,optionally substituted heteroC₅₋₂₀alkyl, optionally substitutedheteroC₅₋₂₀alkenyl, or optionally substituted heteroC₅₋₂₀alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₁₅alkyl, optionallysubstituted C₅₋₁₅alkenyl, optionally substituted C₅₋₁₅alkynyl,optionally substituted heteroC₅₋₁₅alkyl, optionally substitutedheteroC₅₋₁₅alkenyl, or optionally substituted heteroC₅₋₁₅alkynyl. Insome embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅ alkyl, optionally substitutedC₅ alkenyl, optionally substituted C₅ alkynyl, optionally substitutedheteroC₅alkyl, optionally substituted heteroC₅alkenyl, or optionallysubstituted heteroC₅alkynyl. In some embodiments, R⁶ and R⁷ are the samegroup of formula

wherein R^(L) is optionally substituted C₆ alkyl, optionally substitutedC₆ alkenyl, optionally substituted C₆ alkynyl, optionally substitutedheteroC₆alkyl, optionally substituted heteroC₆alkenyl, or optionallysubstituted heteroC₆alkynyl. In some embodiments, R⁶ and R⁷ are the samegroup of formula

wherein R^(L) is optionally substituted C₇ alkyl, optionally substitutedC₇ alkenyl, optionally substituted C₇ alkynyl, optionally substitutedheteroC₇alkyl, optionally substituted heteroC₇alkenyl, or optionallysubstituted heteroC₇alkynyl. In some embodiments, R⁶ and R⁷ are the samegroup of formula

wherein R^(L) is optionally substituted C₈ alkyl, optionally substitutedC₈ alkenyl, optionally substituted C₈ alkynyl, optionally substitutedheteroC₈alkyl, optionally substituted heteroC₈alkenyl, or optionallysubstituted heteroC₈alkynyl. In some embodiments, R⁶ and R⁷ are the samegroup of formula

wherein R^(L) is optionally substituted C₉ alkyl, optionally substitutedC₉ alkenyl, optionally substituted C₉ alkynyl, optionally substitutedheteroC₉alkyl, optionally substituted heteroC₉alkenyl, or optionallysubstituted heteroC₉alkynyl. In some embodiments, R⁶ and R⁷ are the samegroup of formula

wherein R^(L) is optionally substituted C₁₀ alkyl, optionallysubstituted C₁₀alkenyl, optionally substituted C₁₀ alkynyl, optionallysubstituted heteroC₁₀alkyl, optionally substituted heteroC₁₀alkenyl, oroptionally substituted heteroC₁₀alkynyl. In some embodiments, R⁶ and R⁷are the same group of formula

wherein R^(L) is optionally substituted C₁₁ alkyl, optionallysubstituted C₁₁ alkenyl, optionally substituted C₁₁ alkynyl, optionallysubstituted heteroC₁₁alkyl, optionally substituted heteroC₁₁alkenyl, oroptionally substituted heteroC₁₁alkynyl. In some embodiments, R⁶ and R⁷are the same group of formula

wherein R^(L) is optionally substituted C₁₂ alkyl, optionallysubstituted C₁₂ alkenyl, optionally substituted C₁₂ alkynyl, optionallysubstituted heteroC₁₂alkyl, optionally substituted heteroC₁₂alkenyl, oroptionally substituted heteroC₁₂alkynyl. In some embodiments, R⁶ and R⁷are the same group of formula

wherein R^(L) is optionally substituted C₁₃ alkyl, optionallysubstituted C₁₃ alkenyl, optionally substituted C₁₃ alkynyl, optionallysubstituted heteroC₁₃alkyl, optionally substituted heteroC₁₃alkenyl, oroptionally substituted heteroC₁₃alkynyl. In some embodiments, R⁶ and R⁷are the same group of formula

wherein R^(L) is optionally substituted C₁₄ alkyl, optionallysubstituted C₁₄ alkenyl, optionally substituted C₁₄ alkynyl, optionallysubstituted heteroC₁₄alkyl, optionally substituted heteroC₁₄alkenyl, oroptionally substituted heteroC₁₄alkynyl. In some embodiments, R⁶ and R⁷are the same group of formula

wherein R^(L) is optionally substituted C₁₅ alkyl, optionallysubstituted C₁₅ alkenyl, optionally substituted C₁₅ alkynyl, optionallysubstituted heteroC₁₅alkyl, optionally substituted heteroC₁₅alkenyl, oroptionally substituted heteroC₁₅alkynyl.

In some embodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is as defined above and described herein. In someembodiments, R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₋₅₀alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₅₀alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₄₀alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₃₀alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₅alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₂₀alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅₋₁₅alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₅ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₆ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₇ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₈ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₉ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₀ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₁ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₂ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₃ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₄ alkyl. In some embodiments,R⁶ and R⁷ are the same group of formula

wherein R^(L) is optionally substituted C₁₅ alkyl.

As generally defined above, each occurrence of R^(A1) is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, an oxygen protecting group whenattached to an oxygen atom, a sulfur protecting group when attached toan sulfur atom, a nitrogen protecting group when attached to a nitrogenatom, or two R^(A1) groups, together with the nitrogen atom to whichthey are attached, are joined to form an optionally substitutedheterocyclic or optionally substituted heteroaryl ring.

In some embodiments, R^(A1) is hydrogen. In some embodiments, R^(A1) isoptionally substituted alkyl. In some embodiments, R^(A1) is optionallysubstituted alkenyl. In some embodiments, R^(A1) is optionallysubstituted alkynyl. In some embodiments, R^(A1) is optionallysubstituted carbocyclyl. In some embodiments, R^(A1) is optionallysubstituted heterocyclyl. In some embodiments, R^(A1) is optionallysubstituted aryl. In some embodiments, R^(A1) is optionally substitutedheteroaryl. In some embodiments, R^(A1) is an oxygen protecting groupwhen attached to an oxygen atom. In some embodiments, R^(A1) is a sulfurprotecting group when attached to a sulfur atom. In some embodiments,R^(A1) is a nitrogen protecting group when attached to a nitrogen atom.In some embodiments, two R^(A1) groups, together with the nitrogen atomto which they are attached, are joined to form an optionally substitutedheterocyclic or optionally substituted heteroaryl ring.

As generally defined above, each instance of R² is independentlyhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted carbocyclyl,optionally substituted heterocyclyl, optionally substituted aryl,optionally substituted heteroaryl, a nitrogen protecting group, or agroup of the formula (i), (ii), or (iii):

wherein each of R′, Y, R^(P), R^(L) and X is independently as definedabove and described herein.

In some embodiments, R² is hydrogen. In some embodiments, at least oneinstance of R² is hydrogen. In some embodiments, each instance of R² ishydrogen.

In certain embodiments, R² is optionally substituted alkyl; e.g.,optionally substituted C₁₋₆alkyl, optionally substituted C₂₋₆alkyl,optionally substituted C₃₋₆alkyl, optionally substituted C₄₋₆alkyl,optionally substituted C₄₋₅alkyl, or optionally substituted C₃₋₄alkyl.In certain embodiments, at least one instance of R² is optionallysubstituted alkyl; e.g., optionally substituted C₁₋₆alkyl, optionallysubstituted C₂₋₆alkyl, optionally substituted C₃₋₆alkyl, optionallysubstituted C₄₋₆alkyl, optionally substituted C₄₋₅alkyl, or optionallysubstituted C₃₋₄alkyl.

In certain embodiments, R² is optionally substituted alkenyl, e.g.,optionally substituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl,optionally substituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl,or optionally substituted C₃₋₄alkenyl. In certain embodiments, at leastone instance of R² is optionally substituted alkenyl, e.g., optionallysubstituted C₂₋₆alkenyl, optionally substituted C₃₋₆alkenyl, optionallysubstituted C₄₋₆alkenyl, optionally substituted C₄₋₅alkenyl, oroptionally substituted C₃₋₄alkenyl.

In certain embodiments, R² is optionally substituted alkynyl, e.g.,optionally substituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl,optionally substituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl,or optionally substituted C₃₋₄alkynyl. In certain embodiments, at leastone instance of R² is optionally substituted alkynyl, e.g., optionallysubstituted C₂₋₆alkynyl, optionally substituted C₃₋₆alkynyl, optionallysubstituted C₄₋₆alkynyl, optionally substituted C₄₋₅alkynyl, oroptionally substituted C₃₋₄alkynyl.

In certain embodiments, R² is optionally substituted carbocyclyl, e.g.,optionally substituted C₃₋₁₀carbocyclyl, optionally substitutedC₅₋₈carbocyclyl, optionally substituted C₅₋₆carbocyclyl, optionallysubstituted C₅ carbocyclyl, or optionally substituted C₆ carbocyclyl. Incertain embodiments, at least one instance of R² is optionallysubstituted carbocyclyl, e.g., optionally substituted C₃₋₁₀carbocyclyl,optionally substituted C₅₋₈carbocyclyl, optionally substitutedC₅₋₆carbocyclyl, optionally substituted C₅ carbocyclyl, or optionallysubstituted C₆ carbocyclyl.

In certain embodiments, R² is optionally substituted heterocyclyl, e.g.,optionally substituted 3-14 membered heterocyclyl, optionallysubstituted 3-10 membered heterocyclyl, optionally substituted 5-8membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl. In certain embodiments, at leastone instance of R² is optionally substituted heterocyclyl, e.g.,optionally substituted 3-14 membered heterocyclyl, optionallysubstituted 3-10 membered heterocyclyl, optionally substituted 5-8membered heterocyclyl, optionally substituted 5-6 membered heterocyclyl,optionally substituted 5-membered heterocyclyl, or optionallysubstituted 6-membered heterocyclyl.

In certain embodiments, R² is optionally substituted aryl, e.g.,optionally substituted phenyl. In some embodiments, R² is optionallysubstituted phenyl. In some embodiments, R² is substituted phenyl. Insome embodiments, R² is unsubstituted phenyl. In certain embodiments, atleast one instance of R² is optionally substituted aryl, e.g.,optionally substituted phenyl. In some embodiments, at least oneinstance of R² is optionally substituted phenyl. In some embodiments, atleast one instance of R² is substituted phenyl. In some embodiments, atleast one instance of R² is unsubstituted phenyl.

In certain embodiments, R² is optionally substituted heteroaryl, e.g.,optionally substituted 5-14 membered heteroaryl, optionally substituted5-10 membered heteroaryl, optionally substituted 5-6 memberedheteroaryl, optionally substituted 5-membered heteroaryl, or optionallysubstituted 6-membered heteroaryl. In certain embodiments, at least oneinstance of R² is optionally substituted heteroaryl, e.g., optionallysubstituted 5-14 membered heteroaryl, optionally substituted 5-10membered heteroaryl, optionally substituted 5-6 membered heteroaryl,optionally substituted 5-membered heteroaryl, or optionally substituted6-membered heteroaryl.

In some embodiments, R² is a nitrogen protecting group. In someembodiments, at least one R² is a nitrogen protecting group.

In certain embodiments, R² is a group of the formula (i). In certainembodiments, R² is a group of the formula (ii). In certain embodiments,R² is a group of the formula (iii). In certain embodiments, at least oneinstance of R² is a group of the formula (i). In certain embodiments, atleast one instance of R² is a group of the formula (ii). In certainembodiments, at least one instance of R² is a group of the formula(iii).

In certain embodiments, each instance of R² is a group other thanformula (i), (ii), or (iii); in that instance, it follows that at leastone R^(Q) is a group of the formula (i), (ii), or (iii), or at least oneR¹ is a group of formula (iv), and at least one of R⁶ or R⁷ encompassedby R¹ is a group of the formula (i), (ii), or (iii). For example, incertain embodiments, both instances of R² are hydrogen, and thus atleast one R^(Q) is a group of the formula (i), (ii), or (iii), or atleast one R¹ is a group of formula (iv), and at least one of R⁶ or R⁷encompassed by R¹ is a group of the formula (i), (ii), or (iii).

As generally defined above, each instance of R′ is independentlyhydrogen or optionally substituted alkyl. In some embodiments, R′ ishydrogen. In some embodiments, R′ is substituted alkyl. In certainembodiments, at least one instance of R′ is hydrogen. In certainembodiments, at least two instances of R′ is hydrogen. In certainembodiments, each instance of R′ is hydrogen. In certain embodiments, atleast one instance of R′ is optionally substituted alkyl, e.g., methyl.In certain embodiments, at least two instances of R′ is optionallysubstituted alkyl, e.g., methyl. In some embodiments, at least oneinstance of R′ is hydrogen, and at least one instance of R′ isoptionally substituted alkyl. In certain embodiments, one instance of R′is optionally substituted alkyl, and the rest are hydrogen.

As generally defined above, X is O, S, or NR^(X). In some embodiments, Xis O. In some embodiments, X is S. In some embodiments, X is NR^(X),wherein R^(X) is as defined above and described herein.

As generally defined above, R^(X) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group. In some embodiments, R^(X) is hydrogen. Insome embodiments, R^(X) is optionally substituted alkyl. In someembodiments, R^(X) is optionally substituted alkenyl. In someembodiments, R^(X) is optionally substituted alkynyl. In someembodiments, R^(X) is optionally substituted carbocyclyl. In someembodiments, R^(X) is optionally substituted heterocyclyl. In someembodiments, R^(X) is optionally substituted aryl. In some embodiments,R^(X) is optionally substituted heteroaryl. In some embodiments, R^(X)is a nitrogen protecting group.

As generally defined above, Y is O, S, or NR^(Y). In some embodiments, Yis O. In some embodiments, Y is S. In some embodiments, Y is NR^(Y),wherein R^(Y) is as defined above and described herein.

As generally defined above, R^(Y) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group. In some embodiments, R^(Y) is hydrogen. Insome embodiments, R^(Y) is optionally substituted alkyl. In someembodiments, R^(Y) is optionally substituted alkenyl. In someembodiments, R^(Y) is optionally substituted alkynyl. In someembodiments, R^(Y) is optionally substituted carbocyclyl. In someembodiments, R^(Y) is optionally substituted heterocyclyl. In someembodiments, R^(Y) is optionally substituted aryl. In some embodiments,R^(Y) is optionally substituted heteroaryl. In some embodiments, R^(Y)is a nitrogen protecting group.

As generally defined above, R^(P) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to a sulfur atom, or a nitrogenprotecting group when attached to a nitrogen atom. In some embodiments,R^(P) is hydrogen. In some embodiments, R^(P) is optionally substitutedalkyl. In some embodiments, R^(P) is optionally substituted alkenyl. Insome embodiments, R^(P) is optionally substituted alkynyl. In someembodiments, R^(P) is optionally substituted carbocyclyl. In someembodiments, R^(P) is optionally substituted heterocyclyl. In someembodiments, R^(P) is optionally substituted aryl. In some embodiments,R^(P) is optionally substituted heteroaryl. In some embodiments, R^(P)is an oxygen protecting group when attached to an oxygen atom. In someembodiments, R^(P) is a sulfur protecting group when attached to asulfur atom. In some embodiments, R^(P) is a nitrogen protecting groupwhen attached to a nitrogen atom.

As generally defined above, R^(L) is optionally substituted C₁₋₅₀ alkyl,optionally substituted C₂₋₅₀ alkenyl, optionally substituted C₂₋₅₀alkynyl, optionally substituted heteroC₁₋₅₀ alkyl, optionallysubstituted heteroC₂₋₅₀ alkenyl, optionally substituted heteroC₂₋₅₀alkynyl, or a polymer.

In some embodiments, R^(L) is optionally substituted C₁₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₂₋₅₀ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₁ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₀ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₉ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₈ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₇ alkyl. In someembodiments, R^(L) is optionally substituted C₂₋₆ alkyl.

In some embodiments, R^(L) is optionally substituted C₄₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₄₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₁ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₀ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₉ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₈ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₇ alkyl. In someembodiments, R^(L) is optionally substituted C₄₋₆ alkyl.

In some embodiments, R^(L) is optionally substituted C₆₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₆₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₁ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₀ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₉ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₈ alkyl. In someembodiments, R^(L) is optionally substituted C₆₋₇ alkyl.

In some embodiments, R^(L) is optionally substituted C₈₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₈₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₁ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₀ alkyl. In someembodiments, R^(L) is optionally substituted C₈₋₉ alkyl.

In some embodiments, R^(L) is optionally substituted C₉₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₉₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₁ alkyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₀ alkyl.

In some embodiments, R^(L) is optionally substituted C₁₀₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀₋₁₁ alkyl.

In some embodiments, R^(L) is optionally substituted C₁₁₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₁₁₋₁₂ alkyl.

In some embodiments, R^(L) is optionally substituted C₁₂₋₅₀ alkyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₄₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₃₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₂₀ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂₋₁₃ alkyl.

In some embodiments, R^(L) is optionally substituted C₆ alkyl. In someembodiments, R^(L) is optionally substituted C₇ alkyl. In someembodiments, R^(L) is optionally substituted C₈ alkyl. In someembodiments, R^(L) is optionally substituted C₉ alkyl. In someembodiments, R^(L) is optionally substituted C₁₀alkyl. In someembodiments, R^(L) is optionally substituted C₁₁ alkyl. In someembodiments, R^(L) is optionally substituted C₁₂ alkyl. In someembodiments, R^(L) is optionally substituted C₁₃ alkyl. In someembodiments, R^(L) is optionally substituted C₁₄ alkyl. In someembodiments, R^(L) is optionally substituted C₁₅ alkyl. In someembodiments, R^(L) is optionally substituted C₁₆ alkyl. In someembodiments, R^(L) is optionally substituted C₁₇ alkyl. In someembodiments, R^(L) is optionally substituted C₁₈ alkyl. In someembodiments, R^(L) is optionally substituted C₁₉ alkyl. In someembodiments, R^(L) is optionally substituted C₂₀ alkyl.

In some embodiments, for example, in any of the above embodiments, R^(L)is a substituted alkyl group. In some embodiments, R^(L) is anunsubstituted alkyl group. In some embodiments, R^(L) is an optionallysubstituted straight-chain alkyl group. In some embodiments, R^(L) is asubstituted straight-chain alkyl group. In some embodiments, R^(L) is anunsubstituted straight-chain alkyl group. In some embodiments, R^(L) isan optionally substituted branched alkyl group. In some embodiments,R^(L) is a substituted branched alkyl group. In some embodiments, R^(L)is an unsubstituted branched alkyl group.

In certain embodiments, at least one instance of R^(L) is anunsubstituted alkyl. Exemplary unsubstituted alkyl groups include, butare not limited to, —CH₃, —C₂H₅, —C₃H₇, —C₄H₉, —C₅H₁₁, —C₆H₁₃, —C₇H₁₅,—C₈H₁₇, —C₉H₁₉, —C₁₀H₂₁, —C₁₁H₂₃, —C₁₂H₂₅, —C₁₃H₂₇, —C₁₄H₂₉, —C₁₅H₃₁,—C₁₆H₃₃, —C₁₇H₃₅, —C₁₈H₃₇, —C₁₉H₃₉, —C₂₀H₄₁, —C₂₁H₄₃, —C₂₂H₄₅, —C₂₃H₄₇,—C₂₄H₄₉, and —C₂₅H₅₁.

In certain embodiments, at least one instance of R^(L) is a substitutedalkyl. For example, in certain embodiments, at least one instance ofR^(L) is an alkyl substituted with one or more fluorine substituents.Exemplary fluorinated alkyl groups include, but are not limited to:

In some embodiments, R^(L) is optionally substituted C₂₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₂₋₄₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₃₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₂₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₅ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₄ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₃ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₂ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₁ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₋₆ alkenyl.

In some embodiments, R^(L) is optionally substituted C₄₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₄₋₄₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₃₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₂₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₅ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₄ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₃ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₂ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₁ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₄₋₆ alkenyl.

In some embodiments, R^(L) is optionally substituted C₆₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₆₋₄₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₃₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₂₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₅ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₄ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₃ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₂ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₁ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₆₋₇ alkenyl.

In some embodiments, R^(L) is optionally substituted C₈₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₈₋₄₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₃₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₂₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₅ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₄ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₃ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₂ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₁ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₈₋₉ alkenyl.

In some embodiments, R^(L) is optionally substituted C₉₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₉₋₄₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₃₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₂₀ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₅ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₄ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₃ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₂ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₁ alkenyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₀ alkenyl.

In some embodiments, R^(L) is optionally substituted C₁₀₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₄₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₃₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₂₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₉ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₈ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₇ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₆ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₅ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₄ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₃ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₂ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₁ alkenyl.

In some embodiments, R^(L) is optionally substituted C₁₁₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₄₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₃₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₂₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₉ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₈ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₇ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₆ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₅ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₄ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₃ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₂ alkenyl.

In some embodiments, R^(L) is optionally substituted C₁₂₋₅₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₄₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₃₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₂₀ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₉ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₈ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₇ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₆ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₅ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₄ alkenyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₃ alkenyl.

In some embodiments, R^(L) is optionally substituted C₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₀alkenyl. In someembodiments, R^(L) is optionally substituted C₁₁ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₂ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₃ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₄ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₅ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₆ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₇ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₈ alkenyl. In someembodiments, R^(L) is optionally substituted C₁₉ alkenyl. In someembodiments, R^(L) is optionally substituted C₂₀ alkenyl.

In some embodiments, for example, in any of the above embodiments, R^(L)is a substituted alkyl group. In some embodiments, R^(L) is anunsubstituted alkyl group. In some embodiments, R^(L) is an optionallysubstituted straight-chain alkenyl group. In some embodiments, R^(L) isa substituted straight-chain alkenyl group. In some embodiments, R^(L)is an unsubstituted straight-chain alkenyl group. In some embodiments,R^(L) is an optionally substituted branched alkenyl group. In someembodiments, R^(L) is a substituted branched alkenyl group. In someembodiments, R^(L) is an unsubstituted branched alkenyl group.

Exemplary unsubstituted alkenyl group include, but are not limited to:

-   Myristoleic —(CH₂)₇CH═CH(CH₂)₃CH₃,-   Palmitoliec —(CH₂)₇CH═CH(CH₂)₅CH₃,-   Sapienic —(CH₂)₄CH═CH(CH₂)₈CH₃,-   Oleic —(CH₂)₇CH═CH(CH₂)₇CH₃,-   Linoleic —(CH₂)₇CH═CHCH₂CH═CH(CH₂)₄CH₃,-   α-Linolenic —(CH₂)₇CH═CHCH₂CH═CHCH₂CH═CHCH₂CH₃,-   Arachinodonic —(CH₂)₃CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH(CH₂)₄CH₃,-   Eicosapentaenoic —(CH₂)₃CH═CHCH₂CH═CHCH₂CH═CHCH₂H═CHCH₂CH═CHCH₂CH₃,-   Erucic —(CH₂)₁₁CH═CH(CH₂)₇CH₃, and-   Docosahexaenoic    —(CH₂)₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CHCH₂CH═CH—CH₂CH₃.

In some embodiments, wherein R^(L) is defined as a C₆₋₅₀alkyl orC₆₋₅₀alkenyl groups, such groups are meant to encompass lipophilicgroups (also referred to as a “lipid tail”). Lipophilic groups comprisea group of molecules that include fats, waxes, oils, fatty acids, andthe like. Lipid tails present in these lipid groups can be saturated andunsaturated, depending on whether or not the lipid tail comprises doublebonds. The lipid tail can also comprise different lengths, oftencategorized as medium (i.e., with tails between 7-12 carbons, e.g.,C₇₋₁₂ alkyl or C₇₋₁₂ alkenyl), long (i.e., with tails greater than 12carbons and up to 22 carbons, e.g., C₁₃₋₂₂alkyl or C₁₃₋₂₂ alkenyl), orvery long (i.e., with tails greater than 22 carbons, e.g., C₂₃₋₃₀ alkylor C₂₃₋₃₀ alkenyl).

In some embodiments, R^(L) is optionally substituted C₂₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₂₋₄₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₃₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₂₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₅ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₄ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₃ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₂ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₁ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₁₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₋₆ alkynyl.

In some embodiments, R^(L) is optionally substituted C₄₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₄₋₄₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₃₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₂₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₅ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₄ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₃ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₂ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₁ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₁₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₄₋₆ alkynyl.

In some embodiments, R^(L) is optionally substituted C₆₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₆₋₄₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₃₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₂₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₅ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₄ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₃ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₂ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₁ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₁₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₆₋₇ alkynyl.

In some embodiments, R^(L) is optionally substituted C₈₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₈₋₄₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₃₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₂₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₅ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₄ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₃ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₂ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₁ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₁₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₈₋₉ alkynyl.

In some embodiments, R^(L) is optionally substituted C₉₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₉₋₄₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₃₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₂₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₅ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₄ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₃ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₂ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₁ alkynyl. In someembodiments, R^(L) is optionally substituted C₉₋₁₀ alkynyl.

In some embodiments, R^(L) is optionally substituted C₁₀₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₄₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₃₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₂₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₉ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₈ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₇ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₆ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₅ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₄ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₃ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₂ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₀₋₁₁ alkynyl.

In some embodiments, R^(L) is optionally substituted C₁₁₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₄₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₃₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₂₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₉ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₈ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₇ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₆ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₅ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₄ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₃ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₁₋₁₂ alkynyl.

In some embodiments, R^(L) is optionally substituted C₁₂₋₅₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₄₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₃₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₂₀ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₉ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₈ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₇ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₆ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₅ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₄ alkynyl. Insome embodiments, R^(L) is optionally substituted C₁₂₋₁₃ alkynyl.

In some embodiments, R^(L) is optionally substituted C₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₀ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₁ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₂ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₃ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₄ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₅ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₆ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₇ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₈ alkynyl. In someembodiments, R^(L) is optionally substituted C₁₉ alkynyl. In someembodiments, R^(L) is optionally substituted C₂₀ alkynyl.

In some embodiments, for example, in any of the above embodiments, R^(L)is a substituted alkynyl group. In some embodiments, R^(L) is anunsubstituted alkynyl group. In some embodiments, R^(L) is an optionallysubstituted straight-chain alkyl group. In some embodiments, R^(L) is anoptionally substituted straight-chain alkynyl group. In someembodiments, R^(L) is a substituted straight-chain alkynyl group. Insome embodiments, R^(L) is an unsubstituted straight-chain alkynylgroup. In some embodiments, R^(L) is an optionally substituted branchedalkynyl group. In some embodiments, R^(L) is a substituted branchedalkynyl group. In some embodiments, R^(L) is an unsubstituted branchedalkynyl group.

In some embodiments, R^(L) is optionally substituted heteroC₁₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₂alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₁alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₉alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₂₋₈alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₂₋₇alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₂₋₆alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₄₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₂alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₁alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₉alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₄₋₅alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₄₋₇alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₄₋₆alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₆₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₂alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₁alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₉alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₆₋₈alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₆₋₇alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₈₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₂alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₁alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₉alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₉₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₂alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₁alkyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₀alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₂alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₁alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₃alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₂alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₅₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₄₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₃₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₂₀alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₉alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₈alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₇alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₆alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₅alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₄alkyl.In some embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₃alkyl.

In some embodiments, R^(L) is optionally substituted heteroC₆alkyl. Insome embodiments, R^(L) is optionally substituted heteroC₇alkyl. In someembodiments, R^(L) is optionally substituted heteroC₈alkyl. In someembodiments, R^(L) is optionally substituted heteroC₉alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₀alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₁alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₂alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₃alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₄alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₅alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₆alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₇alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₅alkyl. In someembodiments, R^(L) is optionally substituted heteroC₁₉alkyl. In someembodiments, R^(L) is optionally substituted heteroC₂₀alkyl.

In some embodiments, for example, in any of the above embodiments, R^(L)is a substituted heteroalkyl group. In some embodiments, R^(L) is anunsubstituted heteroalkyl group. In some embodiments, R^(L) is anoptionally substituted straight-chain heteroalkyl group. In someembodiments, R^(L) is a substituted straight-chain heteroalkyl group. Insome embodiments, R^(L) is an unsubstituted straight-chain heteroalkylgroup. In some embodiments, R^(L) is an optionally substituted branchedheteroalkyl group. In some embodiments, R^(L) is a substituted branchedheteroalkyl group. In some embodiments, R^(L) is an unsubstitutedbranched heteroalkyl group.

Exemplary unsubstituted heteroalkyl groups include, but are not limitedto:

In some embodiments, R^(L) is optionally substituted heteroC₂₋₅₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₄₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₃₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₂₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₇alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₆alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₅alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₄alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₃alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₂alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₁alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₇alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₆alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₄₋₅₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₄₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₃₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₂₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄17alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄16alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₅alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₄alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₃alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄12alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₁alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₅alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₇alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₆alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₆₋₅₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₄₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₃₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₂₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₇alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₆alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₅alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₄alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₃alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₂alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₁alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₇alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₈₋₅₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₃₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₂₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₇alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₆alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₅alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₄alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₃alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₂alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₁alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₉alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₉₋₅₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₄₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₃₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₂₀alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₈alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₇alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₆alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₅alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₄alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₃alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₂alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₁alkenyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₀alkenyl.

In some embodiments, R^(L) is optionally substitutedheteroC₁₀₋₅₀alkenyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₀₋₄₀alkenyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₀₋₃₀alkenyl. In some embodiments, R^(L)is optionally substituted heteroC₁₀₋₂₀alkenyl. In some embodiments,R^(L) is optionally substituted heteroC₁₀₋₁₉alkenyl. In someembodiments, R^(L) is optionally substituted heteroC₁₀₋₁₈alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₇alkenyl.In some embodiments, R^(L) is optionally substitutedheteroC₁₀₋₁₆alkenyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₀₋₁₅alkenyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₀₋₁₄alkenyl. In some embodiments, R^(L)is optionally substituted heteroC₁₀₋₁₃alkenyl. In some embodiments,R^(L) is optionally substituted heteroC₁₀₋₁₂alkenyl. In someembodiments, R^(L) is optionally substituted heteroC₁₀₋₁₁alkenyl.

In some embodiments, R^(L) is optionally substitutedheteroC₁₁₋₅₀alkenyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₁₋₄₀alkenyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₁₋₃₀alkenyl. In some embodiments, R^(L)is optionally substituted heteroC₁₁₋₂₀alkenyl. In some embodiments,R^(L) is optionally substituted heteroC₁₁₋₁₉alkenyl. In someembodiments, R^(L) is optionally substituted heteroC₁₁₋₁₈alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₇alkenyl.In some embodiments, R^(L) is optionally substitutedheteroC₁₁₋₁₆alkenyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₁₋₁₅alkenyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₁₋₁₄alkenyl. In some embodiments, R^(L)is optionally substituted heteroC₁₁₋₁₃alkenyl. In some embodiments,R^(L) is optionally substituted heteroC₁₁₋₁₂alkenyl.

In some embodiments, R^(L) is optionally substitutedheteroC₁₂₋₅₀alkenyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₂₋₄₀alkenyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₂₋₃₀alkenyl. In some embodiments, R^(L)is optionally substituted heteroC₁₂₋₂₀alkenyl. In some embodiments,R^(L) is optionally substituted heteroC₁₂₋₁₉alkenyl. In someembodiments, R^(L) is optionally substituted heteroC₁₂₋₁₈alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₇alkenyl.In some embodiments, R^(L) is optionally substitutedheteroC₁₂₋₁₆alkenyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₂₋₁₅alkenyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₂₋₁₄alkenyl. In some embodiments, R^(L)is optionally substituted heteroC₁₂₋₁₃alkenyl.

In some embodiments, R^(L) is optionally substituted heteroC₆alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₇alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₈alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₉alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₀alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₁alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₂alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₃alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₄alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₅alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₆alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₇alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₈alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₉alkenyl. Insome embodiments, R^(L) is optionally substituted heteroC₂₀alkenyl.

In some embodiments, for example, in any of the above embodiments, R^(L)is a substituted heteroalkenyl group. In some embodiments, R^(L) is anunsubstituted heteroalkenyl group. In some embodiments, R^(L) is anoptionally substituted straight-chain heteroalkenyl group. In someembodiments, R^(L) is a substituted straight-chain heteroalkenyl group.In some embodiments, R^(L) is an unsubstituted straight-chainheteroalkenyl group. In some embodiments, R^(L) is an optionallysubstituted branched heteroalkenyl group. In some embodiments, R^(L) isa substituted branched heteroalkenyl group. In some embodiments, R^(L)is an unsubstituted branched heteroalkenyl group.

In some embodiments, R^(L) is optionally substituted heteroC₂₋₅₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₄₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₃₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₂₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₆alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₅alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₄alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₃alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₂alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₁alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₁₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₂₋₆alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₄50alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₄₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₃₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₂₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₆alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₅alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄14alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄13alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₂alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₁alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₁₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₅alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₄₋₆alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₆₋₅₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₄₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₃₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₂₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₆alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₅alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₄alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₃alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₂alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₁alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₁₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₆₋₇alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₈₋₅₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₄₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₃₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₂₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₆alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₅alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₄alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₃alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₂alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₁alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₁₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₈₋₉alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₉₋₅₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₄₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₃₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₂₀alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₉alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₈alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₇alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉16alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₅alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₄alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₃alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉12alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₁alkynyl.In some embodiments, R^(L) is optionally substituted heteroC₉₋₁₀alkynyl.

In some embodiments, R^(L) is optionally substitutedheteroC₁₀₋₅₀alkynyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₀₋₄₀alkynyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₀₋₃₀alkynyl. In some embodiments, R^(L)is optionally substituted heteroC₁₀₋₂₀alkynyl. In some embodiments,R^(L) is optionally substituted heteroC₁₀₋₁₉alkynyl. In someembodiments, R^(L) is optionally substituted heteroC₁₀₋₁₈alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₀₋₁₇alkynyl.In some embodiments, R^(L) is optionally substitutedheteroC₁₀₋₁₆alkynyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₀₋₁₅alkynyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₀₋₁₄alkynyl. In some embodiments, R^(L)is optionally substituted heteroC₁₀₋₁₃alkynyl. In some embodiments,R^(L) is optionally substituted heteroC₁₀₋₁₂alkynyl. In someembodiments, R^(L) is optionally substituted heteroC₁₀₋₁₁alkynyl.

In some embodiments, R^(L) is optionally substitutedheteroC₁₁₋₅₀alkynyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₁₋₄₀alkynyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₁₋₃₀alkynyl. In some embodiments, R^(L)is optionally substituted heteroC₁₁₋₂₀alkynyl. In some embodiments,R^(L) is optionally substituted heteroC₁₁₋₁₉alkynyl. In someembodiments, R^(L) is optionally substituted heteroC₁₁₋₁₈alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₁₋₁₇alkynyl.In some embodiments, R^(L) is optionally substitutedheteroC₁₁₋₁₆alkynyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₁₋₁₅alkynyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₁₋₁₄alkynyl. In some embodiments, R^(L)is optionally substituted heteroC₁₁₋₁₃alkynyl. In some embodiments,R^(L) is optionally substituted heteroC₁₁₋₁₂alkynyl.

In some embodiments, R^(L) is optionally substitutedheteroC₁₂₋₅₀alkynyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₂₋₄₀alkynyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₂₋₃₀alkynyl. In some embodiments, R^(L)is optionally substituted heteroC₁₂₋₂₀alkynyl. In some embodiments,R^(L) is optionally substituted heteroC₁₂₋₁₉alkynyl. In someembodiments, R^(L) is optionally substituted heteroC₁₂₋₁₈alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₂₋₁₇alkynyl.In some embodiments, R^(L) is optionally substitutedheteroC₁₂₋₁₆alkynyl. In some embodiments, R^(L) is optionallysubstituted heteroC₁₂₋₁₅alkynyl. In some embodiments, R^(L) isoptionally substituted heteroC₁₂₋₁₄alkynyl. In some embodiments, R^(L)is optionally substituted heteroC₁₂₋₁₃alkynyl.

In some embodiments, R^(L) is optionally substituted heteroC₆alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₇alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₈alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₉alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₀alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₁alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₂alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₃alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₄alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₅alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₆alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₇alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₅alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₁₉alkynyl. Insome embodiments, R^(L) is optionally substituted heteroC₂₀alkynyl.

In some embodiments, for example, in any of the above embodiments, R^(L)is a substituted heteroalkynyl group. In some embodiments, R^(L) is anunsubstituted heteroalkynyl group. In some embodiments, R^(L) is anoptionally substituted straight-chain heteroalkynyl group. In someembodiments, R^(L) is a substituted straight-chain heteroalkynyl group.In some embodiments, R^(L) is an unsubstituted straight-chainheteroalkynyl group. In some embodiments, R^(L) is an optionallysubstituted branched heteroalkynyl group. In some embodiments, R^(L) isa substituted branched heteroalkynyl group. In some embodiments, R^(L)is an unsubstituted branched heteroalkynyl group.

In some embodiments, R^(L) is a polymer. As used herein, a “polymer”, insome embodiments, refers to a compound comprised of at least 3 (e.g., atleast 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeatingcovalently bound structural units. The polymer is in certain embodimentsbiocompatible (i.e., non-toxic). Exemplary polymers include, but are notlimited to, cellulose polymers (e.g., hydroxyethylcellulose,ethylcellulose, carboxymethylcellulose, methylc cellulose,hydroxypropylmethylcellulose (HPMC)), dextran polymers, polymaleic acidpolymers, poly(acrylic acid) polymers, poly(vinylalcohol) polymers,polyvinylpyrrolidone (PVP) polymers, and polyethyleneglycol (PEG)polymers, and combinations thereof.

In some embodiments, R^(L) is a lipophilic, hydrophobic and/or non-polargroup. In some embodiments, R^(L) is a lipophilic group. In someembodiments, R^(L) is a hydrophobic group. In some embodiments, R^(L) isa non-polar group.

In some embodiments, when an R^(L) group is depicted as bisecting acarbon-carbon bond, e.g., of the formula (i), it is understood thatR^(L) may be bonded to either carbon.

In some embodiments, at least one instance of R^(Q), R², R⁶, or R⁷ is agroup of the formula (i), (ii), or (iii). In some embodiments, at leastone instance of R⁶ or R⁷ of R¹ is a group of formula (i), (ii) or (iii).In some embodiments, at least one instance of R⁶ or R⁷ of R¹ is a groupof formula (i). In some embodiments, at least one instance of R⁶ or R⁷of R¹ is a group of formula (i-a). In some embodiments, at least oneinstance of R⁶ or R⁷ of R¹ is a group of formula (i-a1). In someembodiments, at least one instance of R⁶ or R⁷ of R¹ is a group offormula (i-b). In some embodiments, at least one instance of R⁶ or R⁷ ofR¹ is a group of formula (ii). In some embodiments, at least oneinstance of R⁶ or R⁷ of R¹ is a group of formula (iii).

Various combinations of the above embodiments of Formula I arecontemplated herein.

In some embodiments, wherein each instance of Q is O, the compound offormula I is a compound of formula I-a:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In certain embodiments, at least one R¹ is a group of formula (iv). Incertain embodiments, each instance of R¹ is a group of formula (iv). Incertain embodiments, each instance of R² is independently hydrogen oroptionally substituted C₁₋₆alkyl. In certain embodiments, each instanceof R² is hydrogen. In certain embodiments, at least one instance of R²is a group of formula (i). In certain embodiments, at least one instanceof R² is a group of formula (ii). In certain embodiments, at least oneinstance of R² is a group of formula (iii). In certain embodiments, pis 1. In certain embodiments, p is 2. In certain embodiments, p is 3.

In some embodiments, wherein at least one R¹ is a group of formula (iv),a compound of formula I is a compound of formula I-b:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In certain embodiments, each instance of R¹ is a group of formula (iv).In certain embodiments, each instance of R² is independently hydrogen oroptionally substituted C₁₋₆alkyl. In certain embodiments, each instanceof R² is hydrogen. In certain embodiments, at least one instance of R²is a group of formula (i). In certain embodiments, at least one instanceof R² is a group of formula (ii). In certain embodiments, at least oneinstance of R² is a group of formula (iii). In certain embodiments, pis 1. In certain embodiments, p is 2. In certain embodiments, p is 3. Incertain embodiments, L is an optionally substituted alkylene. In certainembodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ isa group of formula (ii). In certain embodiments, R⁶ is a group offormula (iii). In certain embodiments, R⁷ is a group of formula (i). Incertain embodiments, R⁷ is a group of formula (ii). In certainembodiments, R⁷ is a group of formula (iii). In certain embodiments,both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In some embodiments, wherein each instance of R¹ is a group the formula(iv), a compound of Formula I is a compound of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In certain embodiments, each instance of R² is independently hydrogen oroptionally substituted C₁₋₆alkyl. In certain embodiments, each instanceof R² is hydrogen. In certain embodiments, at least one instance of R²is a group of formula (i). In certain embodiments, at least one instanceof R² is a group of formula (ii). In certain embodiments, at least oneinstance of R² is a group of formula (iii). In certain embodiments, pis 1. In certain embodiments, p is 2. In certain embodiments, p is 3. Incertain embodiments, L is an optionally substituted alkylene. In certainembodiments, R⁶ is a group of formula (i). In certain embodiments, R⁶ isa group of formula (ii). In certain embodiments, R⁶ is a group offormula (iii). In certain embodiments, R⁷ is a group of formula (i). Incertain embodiments, R⁷ is a group of formula (ii). In certainembodiments, R⁷ is a group of formula (iii). In certain embodiments,both R⁶ and R⁷ are independently groups of formula (i), (ii), or (iii).

In some embodiments, p=1. In some embodiments, a compound of formula I-cis a compound of formula I-c1:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In certain embodiments, each instance of R² is independently hydrogen oroptionally substituted C₁₋₆alkyl. In certain embodiments, each instanceof R² is hydrogen. In certain embodiments, at least one instance of R²is a group of formula (i). In certain embodiments, at least one instanceof R² is a group of formula (ii). In certain embodiments, at least oneinstance of R² is a group of formula (iii). In certain embodiments, L isan optionally substituted alkylene. In certain embodiments, R⁶ is agroup of formula (i). In certain embodiments, R⁶ is a group of formula(ii). In certain embodiments, R⁶ is a group of formula (iii). In certainembodiments, R⁷ is a group of formula (i). In certain embodiments, R⁷ isa group of formula (ii). In certain embodiments, R⁷ is a group offormula (iii). In certain embodiments, both R⁶ and R⁷ are independentlygroups of formula (i), (ii), or (iii). In some embodiments, R⁶ and R⁷are the same group of formula (i). In some embodiments, R⁶ and R⁷ arethe same group of formula (i-a). In some embodiments, R⁶ and R⁷ are thesame group of formula (i-a1). In some embodiments, R⁶ and R⁷ are thesame group of formula (i-b). In some embodiments, R⁶ and R⁷ are the samegroup of formula (ii). In some embodiments, R⁶ and R⁷ are the same groupof formula (iii).

In some embodiments, each instance of R² is hydrogen. In someembodiments, a compound of formula I-c is a compound of formula I-c2:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In certain embodiments, L is an optionally substituted alkylene. Incertain embodiments, R⁶ is a group of formula (i). In certainembodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶is a group of formula (iii). In certain embodiments, R⁷ is a group offormula (i). In certain embodiments, R⁷ is a group of formula (ii). Incertain embodiments, R⁷ is a group of formula (iii). In certainembodiments, both R⁶ and R⁷ are independently groups of formula (i),(ii), or (iii). In some embodiments, R⁶ and R⁷ are the same group offormula (i). In some embodiments, R⁶ and R⁷ are the same group offormula (i-a). In some embodiments, R⁶ and R⁷ are the same group offormula (i-a1). In some embodiments, R⁶ and R⁷ are the same group offormula (i-b). In some embodiments, R⁶ and R⁷ are the same group offormula (ii). In some embodiments, R⁶ and R⁷ are the same group offormula (iii).

In some embodiments, L is an optionally substituted alkylene. In someembodiments, a compound of formula I-c is a compound of formula I-c3:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, q is an integer between 1 and 10, inclusive. Incertain embodiments, R⁶ is a group of formula (i). In certainembodiments, R⁶ is a group of formula (ii). In certain embodiments, R⁶is a group of formula (iii). In certain embodiments, R⁷ is a group offormula (i). In certain embodiments, R⁷ is a group of formula (ii). Incertain embodiments, R⁷ is a group of formula (iii). In certainembodiments, both R⁶ and R⁷ are independently groups of formula (i),(ii), or (iii). In some embodiments, R⁶ and R⁷ are the same group offormula (i). In some embodiments, R⁶ and R⁷ are the same group offormula (i-a). In some embodiments, R⁶ and R⁷ are the same group offormula (i-a1). In some embodiments, R⁶ and R⁷ are the same group offormula (i-b). In some embodiments, R⁶ and R⁷ are the same group offormula (ii). In some embodiments, R⁶ and R⁷ are the same group offormula (iii).

In some embodiments, a compound of formula I is a compound of formulaI-d:

wherein each of p, R² and R^(L) is independently as defined above anddescribed herein.

In some embodiments, a compound of formula I is a compound of formulaI-e:

wherein each of R² and R^(L) is independently as defined above anddescribed herein.

In some embodiments, a compound of formula I is a compound of formulaI-f:

wherein each of R² and R^(L) is independently as defined above anddescribed herein.

In some embodiments, provided liposomes include a cationic lipiddescribed in WO 2013063468 and in U.S. provisional application entitled“Lipid Formulations for Delivery of Messenger RNA” filed concurrentlywith the present application on even date, both of which areincorporated by reference herein. In some embodiments, a compound offormula I is a compound of formula I-c1-a:

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each R² independently is hydrogen or C₁₋₃ alkyl;    -   each q independently is 2 to 6;    -   each R′ independently is hydrogen or C₁₋₃ alkyl;

and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² independently is hydrogen, methyl or ethyl.In some embodiments, each R² independently is hydrogen or methyl. Insome embodiments, each R² is hydrogen.

In some embodiments, each q independently is 3 to 6. In someembodiments, each q independently is 3 to 5. In some embodiments, each qis 4.

In some embodiments, each R′ independently is hydrogen, methyl or ethyl.In some embodiments, each R′ independently is hydrogen or methyl. Insome embodiments, each R′ independently is hydrogen.

In some embodiments, each R^(L) independently is C₈₋₁₂ alkyl. In someembodiments, each R^(L) independently is n-C₈₋₁₂ alkyl. In someembodiments, each R^(L) independently is C₉₋₁₁ alkyl. In someembodiments, each R^(L) independently is n-C₉₋₁₁ alkyl. In someembodiments, each R^(L) independently is C₁₀alkyl. In some embodiments,each R^(L) independently is n-C₁₀ alkyl.

In some embodiments, each R² independently is hydrogen or methyl; each qindependently is 3 to 5; each R′ independently is hydrogen or methyl;and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q independently is 3 to5; each R′ is hydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, each R² is hydrogen; each q is 4; each R′ ishydrogen; and each R^(L) independently is C₈₋₁₂ alkyl.

In some embodiments, a compound of formula I is a compound of formulaI-g:

wherein each of R^(L) is independently as defined above and describedherein.

In some embodiments, a compound of formula I is a compound of formula X:

or a pharmaceutically acceptable salt thereof, wherein each variable isindependently as defined above and described herein.

In some embodiments, a compound of formula I is a compound of formulaX-1:

or a pharmaceutically acceptable salt thereof, wherein each R² isindependently as defined above and described herein.

In some embodiments, a compound of formula I is

or a pharmaceutically acceptable salt thereof.

Additional examples of cationic lipids suitable for the presentinvention are described in WO 2013063468, which is incorporated byreference herein in its entirety.

Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75th Ed., inside cover, and specificfunctional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5th Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3rd Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various isomeric forms, e.g., enantiomers and/ordiastereomers. For example, the compounds described herein can be in theform of an individual enantiomer, diastereomer or geometric isomer, orcan be in the form of a mixture of stereoisomers, including racemicmixtures and mixtures enriched in one or more stereoisomer. Isomers canbe isolated from mixtures by methods known to those skilled in the art,including chiral high performance liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or preferred isomers canbe prepared by asymmetric syntheses. See, for example, Jacques et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, E. L.Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen,S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L.Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972). Theinvention additionally contemplates compounds as individual isomerssubstantially free of other isomers, and alternatively, as mixtures ofvarious isomers.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C1-6 alkyl” is intended toencompass, C1, C2, C3, C4, C5, C6, C1-6, C1-5, C1-4, C1-3, C1-2, C2-6,C2-5, C2-4, C2-3, C3-6, C3-5, C3-4, C4-6, C4-5, and C5-6 alkyl.

As used herein, “alkyl” refers to a radical of a straight-chain orbranched saturated hydrocarbon group having from 1 to 50 carbon atoms(“C1-50 alkyl”). In some embodiments, an alkyl group has 1 to 40 carbonatoms (“C1-40 alkyl”). In some embodiments, an alkyl group has 1 to 30carbon atoms (“C1-30 alkyl”). In some embodiments, an alkyl group has 1to 20 carbon atoms (“C1-20 alkyl”). In some embodiments, an alkyl grouphas 1 to 10 carbon atoms (“C1-10 alkyl”). In some embodiments, an alkylgroup has 1 to 9 carbon atoms (“C1-9 alkyl”). In some embodiments, analkyl group has 1 to 8 carbon atoms (“C1-8 alkyl”). In some embodiments,an alkyl group has 1 to 7 carbon atoms (“C1-7 alkyl”). In someembodiments, an alkyl group has 1 to 6 carbon atoms (“C1-6 alkyl”). Insome embodiments, an alkyl group has 1 to 5 carbon atoms (“C1-5 alkyl”).In some embodiments, an alkyl group has 1 to 4 carbon atoms (“C1-4alkyl”). In some embodiments, an alkyl group has 1 to 3 carbon atoms(“C1-3 alkyl”). In some embodiments, an alkyl group has 1 to 2 carbonatoms (“C1-2 alkyl”). In some embodiments, an alkyl group has 1 carbonatom (“C1 alkyl”). In some embodiments, an alkyl group has 2 to 6 carbonatoms (“C2-6 alkyl”). Examples of C1-6 alkyl groups include, withoutlimitation, methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3),n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl(C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl(C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkylgroups include n-heptyl (C7), n-octyl (C8) and the like. Unlessotherwise specified, each instance of an alkyl group is independentlyunsubstituted (an “unsubstituted alkyl”) or substituted (a “substitutedalkyl”) with one or more substituents. In certain embodiments, the alkylgroup is an unsubstituted C1-50 alkyl. In certain embodiments, the alkylgroup is a substituted C1-50 alkyl.

As used herein, “heteroalkyl” refers to an alkyl group as defined hereinwhich further includes at least one heteroatom (e.g., 1 to 25, e.g., 1,2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen, boron,silicon, and phosphorus within (i.e., inserted between adjacent carbonatoms of) and/or placed at one or more terminal position(s) of theparent chain. In certain embodiments, a heteroalkyl group refers to asaturated group having from 1 to 50 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC1-50 alkyl”). In certainembodiments, a heteroalkyl group refers to a saturated group having from1 to 40 carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC1-40 alkyl”). In certain embodiments, a heteroalkyl grouprefers to a saturated group having from 1 to 30 carbon atoms and 1 ormore heteroatoms within the parent chain (“heteroC1-30 alkyl”). Incertain embodiments, a heteroalkyl group refers to a saturated grouphaving from 1 to 20 carbon atoms and 1 or more heteroatoms within theparent chain (“heteroC1-20 alkyl”). In certain embodiments, aheteroalkyl group refers to a saturated group having from 1 to 10 carbonatoms and 1 or more heteroatoms within the parent chain (“heteroC1-10alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 9 carbon atoms and 1 or more heteroatoms within the parentchain (“heteroC1-9 alkyl”). In some embodiments, a heteroalkyl group isa saturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC1-8 alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC1-7 alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC1-6 alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC1-5 alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC1-4 alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC1-3alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC1-2 alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC1alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC2-6 alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC1-50 alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC1-50 alkyl.

As used herein, “alkenyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 50 carbon atoms and one ormore carbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C2-50 alkenyl”). In some embodiments, an alkenyl group has 2 to 40carbon atoms (“C2-40 alkenyl”). In some embodiments, an alkenyl grouphas 2 to 30 carbon atoms (“C2-30 alkenyl”). In some embodiments, analkenyl group has 2 to 20 carbon atoms (“C2-20 alkenyl”). In someembodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-10alkenyl”). In some embodiments, an alkenyl group has 2 to 9 carbon atoms(“C2-9 alkenyl”). In some embodiments, an alkenyl group has 2 to 8carbon atoms (“C2-8 alkenyl”). In some embodiments, an alkenyl group has2 to 7 carbon atoms (“C2-7 alkenyl”). In some embodiments, an alkenylgroup has 2 to 6 carbon atoms (“C2-6 alkenyl”). In some embodiments, analkenyl group has 2 to 5 carbon atoms (“C2-5 alkenyl”). In someembodiments, an alkenyl group has 2 to 4 carbon atoms (“C2-4 alkenyl”).In some embodiments, an alkenyl group has 2 to 3 carbon atoms (“C2-3alkenyl”). In some embodiments, an alkenyl group has 2 carbon atoms (“C2alkenyl”). The one or more carbon-carbon double bonds can be internal(such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples ofC2-4 alkenyl groups include, without limitation, ethenyl (C2),1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4),butadienyl (C4), and the like. Examples of C2-6 alkenyl groups includethe aforementioned C2-4 alkenyl groups as well as pentenyl (C5),pentadienyl (C5), hexenyl (C6), and the like. Additional examples ofalkenyl include heptenyl (C7), octenyl (C8), octatrienyl (C8), and thelike. Unless otherwise specified, each instance of an alkenyl group isindependently unsubstituted (an “unsubstituted alkenyl”) or substituted(a “substituted alkenyl”) with one or more substituents. In certainembodiments, the alkenyl group is an unsubstituted C2-50 alkenyl. Incertain embodiments, the alkenyl group is a substituted C2-50 alkenyl.

As used herein, “heteroalkenyl” refers to an alkenyl group as definedherein which further includes at least one heteroatom (e.g., 1 to 25,e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen,boron, silicon, and phosphorus within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkenyl group refersto a group having from 2 to 50 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC2-50alkenyl”). In certain embodiments, a heteroalkenyl group refers to agroup having from 2 to 40 carbon atoms, at least one double bond, and 1or more heteroatoms within the parent chain (“heteroC2-40 alkenyl”). Incertain embodiments, a heteroalkenyl group refers to a group having from2 to 30 carbon atoms, at least one double bond, and 1 or moreheteroatoms within the parent chain (“heteroC2-30 alkenyl”). In certainembodiments, a heteroalkenyl group refers to a group having from 2 to 20carbon atoms, at least one double bond, and 1 or more heteroatoms withinthe parent chain (“heteroC2-20 alkenyl”). In certain embodiments, aheteroalkenyl group refers to a group having from 2 to 10 carbon atoms,at least one double bond, and 1 or more heteroatoms within the parentchain (“heteroC2-10 alkenyl”). In some embodiments, a heteroalkenylgroup has 2 to 9 carbon atoms at least one double bond, and 1 or moreheteroatoms within the parent chain (“heteroC2-9 alkenyl”). In someembodiments, a heteroalkenyl group has 2 to 8 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC2-8 alkenyl”). In some embodiments, a heteroalkenyl group has 2to 7 carbon atoms, at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC2-7 alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC2-6alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC2-5 alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms. at least one double bond,and for 2 heteroatoms within the parent chain (“heteroC2-4 alkenyl”). Insome embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC2-3 alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double, bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC2-6 alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC2-50alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC2-50 alkenyl.

As used herein, “alkynyl” refers to a radical of a straight-chain orbranched hydrocarbon group having from 2 to 50 carbon atoms and one ormore carbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) andoptionally one or more double bonds (e.g., 1, 2, 3, or 4 double bonds)(“C2-50 alkynyl”). An alkynyl group that has one or more triple bondsand one or more double bonds is also referred to as an “ene-yne”. Insome embodiments, an alkynyl group has 2 to 40 carbon atoms (“C2-40alkynyl”). In some embodiments, an alkynyl group has 2 to 30 carbonatoms (“C2-30 alkynyl”). In some embodiments, an alkynyl group has 2 to20 carbon atoms (“C2-20 alkynyl”). In some embodiments, an alkynyl grouphas 2 to 10 carbon atoms (“C2-10 alkynyl”). In some embodiments, analkynyl group has 2 to 9 carbon atoms (“C2-9 alkynyl”). In someembodiments, an alkynyl group has 2 to 8 carbon atoms (“C2-8 alkynyl”).In some embodiments, an alkynyl group has 2 to 7 carbon atoms (“C2-7alkynyl”). In some embodiments, an alkynyl group has 2 to 6 carbon atoms(“C2-6 alkynyl”). In some embodiments, an alkynyl group has 2 to 5carbon atoms (“C2-5 alkynyl”). In some embodiments, an alkynyl group has2 to 4 carbon atoms (“C2-4 alkynyl”). In some embodiments, an alkynylgroup has 2 to 3 carbon atoms (“C2-3 alkynyl”). In some embodiments, analkynyl group has 2 carbon atoms (“C2 alkynyl”). The one or morecarbon-carbon triple bonds can be internal (such as in 2-butynyl) orterminal (such as in 1-butynyl). Examples of C2-4 alkynyl groupsinclude, without limitation, ethynyl (C2), 1-propynyl (C3), 2-propynyl(C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Examples of C2-6alkenyl groups include the aforementioned C2-4 alkynyl groups as well aspentynyl (C5), hexynyl (C6), and the like. Additional examples ofalkynyl include heptynyl (C7), octynyl (C8), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted C2-50 alkynyl. Incertain embodiments, the alkynyl group is a substituted C2-50 alkynyl.

As used herein, “heteroalkynyl” refers to an alkynyl group as definedherein which further includes at least one heteroatom (e.g., 1 to 25,e.g., 1, 2, 3, or 4 heteroatoms) selected from oxygen, sulfur, nitrogen,boron, silicon, and phosphorus within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkynyl group refersto a group having from 2 to 50 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC2-50alkynyl”). In certain embodiments, a heteroalkynyl group refers to agroup having from 2 to 40 carbon atoms, at least one triple bond, and 1or more heteroatoms within the parent chain (“heteroC2-40 alkynyl”). Incertain embodiments, a heteroalkynyl group refers to a group having from2 to 30 carbon atoms, at least one triple bond, and 1 or moreheteroatoms within the parent chain (“heteroC2-30 alkynyl”). In certainembodiments, a heteroalkynyl group refers to a group having from 2 to 20carbon atoms, at least one triple bond, and 1 or more heteroatoms withinthe parent chain (“heteroC2-20 alkynyl”). In certain embodiments, aheteroalkynyl group refers to a group having from 2 to 10 carbon atoms,at least one triple bond, and 1 or more heteroatoms within the parentchain (“heteroC2-10 alkynyl”). In some embodiments, a heteroalkynylgroup has 2 to 9 carbon atoms, at least one triple bond, and 1 or moreheteroatoms within the parent chain (“heteroC2-9 alkynyl”). In someembodiments, a heteroalkynyl group has 2 to 8 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC2-8 alkynyl”). In some embodiments, a heteroalkynyl group has 2to 7 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC2-7 alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC2-6alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC2-5 alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and for 2 heteroatoms within the parent chain (“heteroC2-4 alkynyl”). Insome embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC2-3 alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond. and 1 or 2 heteroatomswithin the parent chain (“heteroC2-6 alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC2-50alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC2-50 alkynyl.

As used herein, “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 10 ring carbonatoms (“C3-10 carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 8 ringcarbon atoms (“C3-8 carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 7 ring carbon atoms (“C3-7 carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 6 ring carbon atoms (“C3-6carbocyclyl”). In some embodiments, a carbocyclyl group has 4 to 6 ringcarbon atoms (“C4-6 carbocyclyl”). In some embodiments, a carbocyclylgroup has 5 to 6 ring carbon atoms (“C5-6 carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 10 ring carbon atoms (“C5-10carbocyclyl”). Exemplary C3-6 carbocyclyl groups include, withoutlimitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4),cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl(C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. ExemplaryC3-8 carbocyclyl groups include, without limitation, the aforementionedC3-6 carbocyclyl groups as well as cycloheptyl (C7), cycloheptenyl (C7),cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8),cyclooctenyl (C8), bicyclo[2.2.1]heptanyl (C7), bicyclo[2.2.2]octanyl(C8), and the like. Exemplary C3-10 carbocyclyl groups include, withoutlimitation, the aforementioned C3-8 carbocyclyl groups as well ascyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl(C10), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C10),spiro[4.5]decanyl (C10), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C3-10carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C3-10 carbocyclyl.

In some embodiments, “carbocyclyl” or “carbocyclic” is referred to as a“cycloalkyl”, i.e., a monocyclic, saturated carbocyclyl group havingfrom 3 to 10 ring carbon atoms (“C3-10 cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 6 ringcarbon atoms (“C3-6, cycloalkyl”). In some embodiments, a cycloalkylgroup has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C5-10 cycloalkyl”). Examples of C5-6 cycloalkyl groupsinclude cyclopentyl (C5) and cyclohexyl (C5). Examples of C3-6cycloalkyl groups include the aforementioned C5-6 cycloalkyl groups aswell as cyclopropyl (C3) and cyclobutyl (C4). Examples of C3-8cycloalkyl groups include the aforementioned C3-6 cycloalkyl groups aswell as cycloheptyl (C7) and cyclooctyl (C8). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with one or more substituents. In certainembodiments, the cycloalkyl group is an unsubstituted C3-10 cycloalkyl.In certain embodiments, the cycloalkyl group is a substituted C3-10cycloalkyl.

As used herein, “heterocyclyl” or “heterocyclic” refers to a radical ofa 3- to 14-membered non-aromatic ring system having ring carbon atomsand 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, and phosphorus (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)). and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1 or more (e.g.,1, 2, 3, or 4) ring heteroatoms, wherein each heteroatom isindependently selected from oxygen, sulfur, nitrogen, boron, silicon,and phosphorus (“5-10 membered heterocyclyl”). In some embodiments, aheterocyclyl group is a 5-8 membered non-aromatic ring system havingring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ring heteroatoms,wherein each heteroatom is independently selected from oxygen, sulfur,nitrogen, boron, silicon, and phosphorus (“5-8 membered heterocyclyl”).In some embodiments, a heterocyclyl group is a 5-6 membered non-aromaticring system having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4)ring heteroatoms, wherein each heteroatom is independently selected fromoxygen, sulfur, nitrogen, boron, silicon, and phosphorus (“5-6 memberedheterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1or more (e.g., 1, 2, or 3) ring heteroatoms selected from oxygen,sulfur, nitrogen, boron, silicon, and phosphorus. In some embodiments,the 5-6 membered heterocyclyl has 1 or 2 ring heteroatoms selected fromoxygen, sulfur, nitrogen, boron, silicon, and phosphorus. In someembodiments, the 5-6 membered heterocyclyl has 1 ring heteroatomselected from oxygen, sulfur, nitrogen, boron, silicon, and phosphorus.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary4-membered heterocyclyl groups containing 1 heteroatom include, withoutlimitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-memberedheterocyclyl groups containing 1 heteroatom include, without limitation.tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl,dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione.Exemplary 5-membered heterocyclyl groups containing 2 heteroatomsinclude, without limitation, dioxolanyl, oxathiolanyl and dithiolanyl.Exemplary 5-membered heterocyclyl groups containing 3 heteroatomsinclude, without limitation, triazolinyl, oxadiazolinyl, andthiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing 1heteroatom include, without limitation, piperidinyl, tetrahydropyranyl,dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, piperazinyl,morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclylgroups containing 2 heteroatoms include, without limitation,triazinanyl. Exemplary 7-membered heterocyclyl groups containing 1heteroatom include, without limitation, azepanyl, oxepanyl andthiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo-[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

As used herein, “aryl” refers to a radical of a monocyclic or polycyclic(e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6,10, or 14π electrons shared in a cyclic array) having 6-14 ring carbonatoms and zero heteroatoms provided in the aromatic ring system (“C6-14aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C6aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C14 aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C6-14 aryl. In certainembodiments, the aryl group is a substituted C6-14 aryl.

As used herein, “heteroaryl” refers to a radical of a 5-14 memberedmonocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromaticring system (e.g., having 6, 10, or 14 π electrons shared in a cyclicarray) having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4 ringheteroatoms) ring heteroatoms provided in the aromatic ring system,wherein each heteroatom is independently selected from oxygen, sulfur,nitrogen, boron, silicon, and phosphorus (“5-14 membered heteroaryl”).In heteroaryl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits.Heteroaryl polycyclic ring systems can include one or more heteroatomsin one or both rings. “Heteroaryl” includes ring systems wherein theheteroaryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the point of attachment is on theheteroaryl ring, and in such instances, the number of ring memberscontinue to designate the number of ring members in the heteroaryl ringsystem. “Heteroaryl” also includes ring systems wherein the heteroarylring, as defined above, is fused with one or more aryl groups whereinthe point of attachment is either on the aryl or heteroaryl ring, and insuch instances, the number of ring members designates the number of ringmembers in the fused polycyclic (aryl/heteroaryl) ring system.Polycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, and phosphorus (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, and phosphorus (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1 or more (e.g., 1, 2, 3, or 4) ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from oxygen, sulfur, nitrogen,boron, silicon, and phosphorus (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1 or more (e.g., 1, 2, or3) ring heteroatoms selected from oxygen, sulfur, nitrogen, boron,silicon, and phosphorus. In some embodiments, the 5-6 memberedheteroaryl has 1 or 2 ring heteroatoms selected from oxygen, sulfur,nitrogen, boron, silicon, and phosphorus. In some embodiments, the 5-6membered heteroaryl has 1 ring heteroatom selected from oxygen, sulfur,nitrogen, boron, silicon, and phosphorus. Unless otherwise specified,each instance of a heteroaryl group is independently unsubstituted (an“unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”)with one or more substituents. In certain embodiments, the heteroarylgroup is an unsubstituted 5-14 membered heteroaryl. In certainembodiments, the heteroaryl group is a substituted 5-14 memberedheteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation. pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aromatic groups (e.g., arylor heteroaryl moieties) as herein defined.

As used herein, the term “saturated” refers to a ring moiety that doesnot contain a double or triple bond, i.e., the ring contains all singlebonds.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl,

alkynylene is the divalent moiety of alkynyl, heteroalkylene is thedivalent moiety of heteroalkyl, heteroalkenylene is the divalent moietyof heteroalkenyl, heteroalkynylene is the divalent moiety ofheteroalkynyl, carbocyclylene is the divalent moiety of carbocyclyl,heterocyclylene is the divalent moiety of heterocyclyl, arylene is thedivalent moiety of aryl, and heteroarylene is the divalent moiety ofheteroaryl.

As understood from the above, alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl groups, as defined herein, are, in certain embodiments,optionally substituted. Optionally substituted refers to a group whichmay be substituted or unsubstituted (e.g., “substituted” or“unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl,“substituted” or “unsubstituted” alkynyl, “substituted” or“unsubstituted” heteroalkyl, “substituted” or “unsubstituted”heteroalkenyl, “substituted” or “unsubstituted” heteroalkynyl.“substituted” or “unsubstituted” carbocyclyl. “substituted” or“unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or“substituted” or “unsubstituted” heteroaryl group). In general, the term“substituted” means that at least one hydrogen present on a group isreplaced with a permissible substituent, e.g., a substituent which uponsubstitution results in a stable compound, e.g., a compound which doesnot spontaneously undergo transformation such as by rearrangement,cyclization, elimination, or other reaction. Unless otherwise indicated,a “substituted” group has a substituent at one or more substitutablepositions of the group, and when more than one position in any givenstructure is substituted, the substituent is either the same ordifferent at each position. The term “substituted” is contemplated toinclude substitution with all permissible substituents of organiccompounds, any of the substituents described herein that results in theformation of a stable compound. The present invention contemplates anyand all such combinations in order to arrive at a stable compound. Forpurposes of this invention, heteroatoms such as nitrogen may havehydrogen substituents and/or any suitable substituent as describedherein which satisfy the valencies of the heteroatoms and results in theformation of a stable moiety.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO2, —N3, —SO2H, —SO3H, —OH, —ORaa, —ON(Rbb)2, —N(Rbb)2,—N(Rbb)3+X—, —N(ORcc)Rbb, —SeH, —SeRaa, —SH, —SRaa, —SSRcc, —C(═O)Raa,—CO2H, —CHO, —C(ORcc)2, —CO2Raa, —OC(═O)Raa, —OCO2Raa, —C(═O)N(Rbb)2,—OC(═O)N(Rbb)2, —NRbbC(═O)Raa, —NRbbCO2Raa, —NRbbC(═O)N(Rbb)2,—C(═NRbb)Raa, —C(═NRbb)ORaa, —OC(═NRbb)Raa, —OC(═NRbb)ORaa,—C(═NRbb)N(Rbb)2, —OC(═NRbb)N(Rbb)2, —NRbbC(═NRbb)N(Rbb)2,—C(═O)NRbbSO2Raa, —NRbbSO2Raa, —SO2N(Rbb)2, —SO2Raa, —SO2ORaa, —OSO2Raa,—S(═O)Raa, —OS(═O)Raa, —Si(Raa)3 —OSi(Raa)3 —C(═S)N(Rbb)2, —C(═O)SRaa,—C(═S)SRaa, —SC(═S)SRaa, —SC(═O)SRaa, —OC(═O)SRaa, —SC(═O)ORaa,—SC(═O)Raa, —P(═O)2Raa, —OP(═O)2Raa, —P(═O)(Raa)2, —OP(═O)(Raa)2,—OP(═O)(ORcc)2, —P(═O)2N(Rbb)2, —OP(═O)2N(Rbb)2, —P(═O)(NRbb)2,—OP(═O)(NRbb)2, —NRbbP(═O)(ORcc)2, —NRbbP(═O)(NRbb)2, —P(Rcc)2,—P(Rcc)3, —OP(Rcc)2, —OP(Rcc)3, —B(Raa)2, —B(ORcc)2, —BRaa(ORcc), C1-50alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-14 carbocyclyl, 3-14 memberedheterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, wherein eachalkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroarylis independently substituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(Rbb)2, ═NNRbbC(═O)Raa, ═NNRbbC(═O)ORaa, ═NNRbbS(═O)2Raa,═NRbb, or ═NORcc;

each instance of Raa is, independently, selected from C1-50 alkyl, C2-50alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl,C6-14 aryl, and 5-14 membered heteroaryl, or two Raa groups are joinedto form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups;

each instance of Rbb is, independently, selected from hydrogen, —OH,—ORaa, —N(Rcc)2, —CN, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa,—C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO20Rcc, —SORaa,—C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2,—P(═O)2N(Rcc)2, —P(═O)(NRcc)2, C1-50 alkyl, C2-50 alkenyl, C2-50alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and5-14 membered heteroaryl, or two Rbb groups, together with theheteroatom to which they are attached, form a 3-14 membered heterocyclylor 5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 Rdd groups;

each instance of Rcc is, independently, selected from hydrogen, C1-50alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-14 memberedheterocyclyl, C6-14 aryl, and 5-14 membered heteroaryl, or two Rccgroups, together with the heteroatom to which they are attached, form a3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rddgroups;

each instance of Rdd is, independently, selected from halogen, —CN,—NO2, —N3, —SO2H, —SO3H, —OH, —ORee, —ON(Rff)2, —N(Rff)2, —N(Rff)3+X—,—N(ORee)Rff, —SH, —SRee, —SSRee, —C(═O)Ree, —CO2H, —CO2Ree, —OC(═O)Ree,—OCO2Ree, —C(═O)N(Rff)2, —OC(═O)N(Rff)2, —NRffC(═O)Ree, —NRffCO2Ree,—NRffC(═O)N(Rff)2, —C(═NRff)ORee, —OC(═NRff)Ree, —OC(═NRff)ORee,—C(═NRff)N(Rff)2, —OC(═NRff)N(Rff)2, —NRffC(═NRff)N(Rff)2, —NRffSO2Ree,—SO2N(Rff)2, —SO2Ree, —SO2ORee, —OSO2Ree, —S(═O)Ree, —Si(Ree)3,—OSi(Ree)3, —C(═S)N(Rff)2, —C(═O)SRee, —C(═S)SRee, —SC(═S)SRee,—P(═O)2Ree, —P(═O)(Ree)2, —OP(═O)(Ree)2, —OP(═O)(ORee)2, C1-50 alkyl,C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-10 memberedheterocyclyl, C6-10 aryl, 5-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups, or twogeminal Rdd substituents can be joined to form ═O or ═S;

each instance of Ree is, independently, selected from C1-50 alkyl, C2-50alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, C6-10 aryl, 3-10 memberedheterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently substituted with 0, 1, 2, 3, 4, or 5 Rgg groups;

each instance of Rff is, independently, selected from hydrogen, C1-50alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, 3-10 memberedheterocyclyl, C6-10 aryl and 5-10 membered heteroaryl, or two Rffgroups, together with the heteroatom to which they are attached, form a3-14 membered heterocyclyl or 5-14 membered heteroaryl ring, whereineach alkyl, alkenyl, alkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 Rgggroups; and

each instance of Rgg is, independently, halogen, —CN, —NO2, —N3, —SO2H,—SO3H, —OH, —OC1-50 alkyl, —ON(C1-50 alkyl)2, —N(C1-50 alkyl)2, —N(C1-50alkyl)3+X—, —NH(C1-50 alkyl)2+X—, —NH2(C1-50 alkyl)+X—, —NH3+X—,—N(OC1-50 alkyl)(C1-50 alkyl), —N(OH)(C1-50 alkyl), —NH(OH), —SH,—SC1-50 alkyl, —SS(C1-50 alkyl), —C(═O)(C1-50 alkyl), —CO2H, —CO2(C1-50alkyl), —OC(═O)(C1-50 alkyl), —OCO2(C1-50 alkyl), —C(═O)NH2,—C(═O)N(C1-50 alkyl)2, —OC(═O)NH(C1-50 alkyl), —NHC(═O)(C1-50 alkyl),—N(C1-50 alkyl)C(═O)(C1-50 alkyl), —NHCO2(C1-50 alkyl), —NHC(═O)N(C1-50alkyl)2, —NHC(═O)NH(C1-50 alkyl), —NHC(═O)NH2, —C(═NH)O(C1-50 alkyl),—OC(═NH)(C1-50 alkyl), —OC(═NH)OC1-50 alkyl, —C(═NH)N(C1-50 alkyl)2,—C(═NH)NH(C1-50 alkyl), —C(═NH)NH2, —OC(═NH)N(C1-50alkyl)2,—OC(NH)NH(C1-50 alkyl), —OC(NH)NH2, —NHC(NH)N(C1-50 alkyl)2,—NHC(═NH)NH2, —NHSO2 (C1-50 alkyl), —SO2N (C1-50 alkyl)2, —SO2NH (C1-50alkyl), —SO2NH2, —SO2C1-50 alkyl, —SO2OC1-50 alkyl, —OSO2C1-6 alkyl,—SOC1-6 alkyl, —Si(C1-50 alkyl)3, —OSi(C1-6 alkyl)3 —C(═S)N(C1-50alkyl)2, C(═S)NH(C1-50 alkyl), C(═S)NH2, —C(═O)S(C1-6 alkyl),—C(═S)SC1-6 alkyl, —SC(═S)SC1-6 alkyl, —P(═O)2(C1-50 alkyl),—P(═O)(C1-50 alkyl)2, —OP(═O)(C1-50 alkyl)2, —OP(═O)(OC1-50 alkyl)2,C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10 carbocyclyl, C6-10aryl, 3-10 membered heterocyclyl, 5-10 membered heteroaryl; or twogeminal Rgg substituents can be joined to form ═O or ═S;

wherein X— is a counterion.

As used herein, the term “halo” or “halogen” refers to fluorine (fluoro,—F), chlorine (chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

As used herein, a “counterion” is a negatively charged group associatedwith a positively charged quarternary amine in order to maintainelectronic neutrality. Exemplary counterions include halide ions (e.g.,F—, Cl—, Br—, I—), NO3-, ClO4-, OH—, H2PO4-, HSO4-, sulfonate ions(e.g., methansulfonate, trifluoromethanesulfonate, p-toluenesulfonate,benzenesulfonate, 10-camphor sulfonate, naphthalene-2-sulfonate,naphthalene-1-sulfonic acid-5-sulfonate, ethan-1-sulfonicacid-2-sulfonate, and the like), and carboxylate ions (e.g., acetate,ethanoate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, and the like).

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa,—C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa,—C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2,—C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2,—P(═O)(NRcc)2, C1-50 alkyl, C2-50 alkenyl, C2-50 alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 memberedheteroaryl, or two Rcc groups, together with the N atom to which theyare attached, form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently substituted with 0,1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rdd are asdefined above.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quarternary nitrogenatoms. Exemplary nitrogen atom substitutents include, but are notlimited to, hydrogen, —OH, —ORaa, —N(Rcc)2, —CN, —C(═O)Raa,—C(═O)N(Rcc)2, —CO2Raa, —SO2Raa, —C(═NRbb)Raa, —C(═NRcc)ORaa,—C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc, —SO2ORcc, —SORaa, —C(═S)N(Rcc)2,—C(═O)SRcc, —C(═S)SRcc, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)2N(Rcc)2,—P(═O)(NRcc)2, C1-10 alkyl, C1-10 perhaloalkyl, C2-10 alkenyl, C2-10alkynyl, C3-10 carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and5-14 membered heteroaryl, or two Rcc groups, together with the nitrogenatom to which they are attached, form a 3-14 membered heterocyclyl or5-14 membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 Rdd groups, and wherein Raa, Rbb,Rcc and Rdd are as defined above.

In certain embodiments, the substituent present on a nitrogen atom is anitrogen protecting group (also referred to as an amino protectinggroup). Nitrogen protecting groups include, but are not limited to, —OH,—ORaa, —N(Rcc)2, —C(═O)Raa, —C(═O)N(Rcc)2, —CO2Raa, —SO2Raa,—C(═NRcc)Raa, —C(═NRcc)ORaa, —C(═NRcc)N(Rcc)2, —SO2N(Rcc)2, —SO2Rcc,—SO2ORcc, —SORaa, —C(═S)N(Rcc)2, —C(═O)SRcc, —C(═S)SRcc, C1-10 alkyl(e.g., aralkyl, heteroaralkyl), C2-10 alkenyl, C2-10 alkynyl, C3-10carbocyclyl, 3-14 membered heterocyclyl, C6-14 aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, carbocyclyl,heterocyclyl, aralkyl, aryl, and heteroaryl is independently substitutedwith 0, 1, 2, 3, 4 or 5 Rdd groups, and wherein Raa, Rbb, Rcc and Rddare as defined herein. Nitrogen protecting groups are well known in theart and include those described in detail in Protecting Groups inOrganic Synthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, JohnWiley & Sons, 1999, incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)Raa) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitrophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g., —C(═O)ORaa)include, but are not limited to, methyl carbamate, ethyl carbamate,9-fluorenylmethyl carbamate (Fmoc), 9-(2-sulfo)fluorenylmethylcarbamate, 9-(2,7-dibromo)fluoroenylmethyl carbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC), 1-adamantyl carbamate (Adoc), vinylcarbamate (Voc), allyl carbamate (Alloc), 1-isopropylallyl carbamate(Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate (Noc),8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methyl sulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g., —S(═O)2Raa)include, but are not limited to, p-toluenesulfonamide (Ts),benzenesulfonamide, 2,3,6,-trimethyl-4-methoxybenzenesulfonamide (Mtr),2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pmc), methanesulfonamide(Ms), β-trimethyl silylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine, N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N-5-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N-5-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to as a hydroxyl protectinggroup). Oxygen protecting groups include, but are not limited to, —Raa,—N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, —C(═O)N(Rbb)2, —C(═NRbb)Raa,—C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, Si(Raa)3, —P(Rcc)2,—P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2, —P(═O)2N(Rbb)2, and—P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are as defined herein. Oxygenprotecting groups are well known in the art and include those describedin detail in Protecting Groups in Organic Synthesis, T. W. Greene and P.G. M. Wuts, 3rd edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethyl silylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, a-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodisulfuran-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), alkyl methyl carbonate,9-fluorenylmethyl carbonate (Fmoc), alkyl ethyl carbonate, alkyl2,2,2-trichloroethyl carbonate (Troc), 2-(trimethylsilyl)ethyl carbonate(TMSEC), 2-(phenylsulfonyl) ethyl carbonate (Psec),2-(triphenylphosphonio) ethyl carbonate (Peoc), alkyl isobutylcarbonate, alkyl vinyl carbonate alkyl allyl carbonate, alkylp-nitrophenyl carbonate, alkyl benzyl carbonate, alkyl p-methoxybenzylcarbonate, alkyl 3,4-dimethoxybenzyl carbonate, alkyl o-nitrobenzylcarbonate, alkyl p-nitrobenzyl carbonate, alkyl S-benzyl thiocarbonate,4-ethoxy-1-naphthyl carbonate, methyl dithiocarbonate, 2-iodobenzoate,4-azidobutyrate, 4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, a-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts).

In certain embodiments, the substituent present on an sulfur atom is ansulfur protecting group (also referred to as a thiol protecting group).Sulfur protecting groups include, but are not limited to, —Raa,—N(Rbb)2, —C(═O)SRaa, —C(═O)Raa, —CO2Raa, C(═O)N(Rbb)2, —C(═NRbb)Raa,—C(═NRbb)ORaa, —C(═NRbb)N(Rbb)2, —S(═O)Raa, —SO2Raa, —Si(Raa)3,—P(Rcc)2, —P(Rcc)3, —P(═O)2Raa, —P(═O)(Raa)2, —P(═O)(ORcc)2,—P(═O)2N(Rbb)2, and —P(═O)(NRbb)2, wherein Raa, Rbb, and Rcc are asdefined herein. Sulfur protecting groups are well known in the art andinclude those described in detail in Protecting Groups in OrganicSynthesis, T. W. Greene and P. G. M. Wuts, 3rd edition, John Wiley &Sons, 1999, incorporated herein by reference.

As used herein, a “leaving group” is an art-understood term referring toa molecular fragment that departs with a pair of electrons inheterolytic bond cleavage, wherein the molecular fragment is an anion orneutral molecule. See, for example, Smith, March's Advanced OrganicChemistry 6th ed. (501-502). Exemplary leaving groups include, but arenot limited to, halo (e.g., chloro, bromo, iodo) and sulfonylsubstituted hydroxyl groups (e.g., tosyl, mesyl, besyl).

Other Definitions

As used herein, use of the phrase “at least one instance” refers to oneinstance, but also encompasses more than one instance, e.g., forexample, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 instances, and up to 100instances.

As used herein, a “polymer” refers to a compound comprised of at least 3(e.g., at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.) repeatingcovalently bound structural units.

“Attached” refers to the covalent attachment of a group.

As used herein, “lipophilic” refers to the ability of a group todissolve in fats, oils, lipids, and lipophilic non-polar solvents suchas hexane or toluene. In general, a lipophilic group refers to anunsubstituted n-alkyl or unsubstituted n-alkenyl group having 6 to 50carbon atoms, e.g., 6 to 40, 6 to 30, 6 to 20, 8 to 20, 8 to 19, 8 to18, 8 to 17, 8 to 16, or 8 to 15 carbon atoms.

As used herein, the term “salt” or “pharmaceutically acceptable salt”refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al., describes pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences (1977) 66:1-19. Pharmaceuticallyacceptable salts of the compounds of this invention include thosederived from suitable inorganic and organic acids and bases. Examples ofpharmaceutically acceptable, nontoxic acid addition salts are salts ofan amino group formed with inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate.digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N+(C1-4alkyl)4 salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium. quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, sulfonate and aryl sulfonate.Further pharmaceutically acceptable salts include salts formed from thequaternization of an amine using an appropriate electrophile, e.g., analkyl halide, to form a quarternized alkylated amino salt.

Second or Additional Cationic Lipids

In some embodiments, liposomes may comprise a second or additionalcationic lipid. As used herein, the phrase “cationic lipid” refers toany of a number of lipid species that have a net positive charge at aselected pH, such as physiological pH. Several cationic lipids have beendescribed in the literature, many of which are commercially available.Particularly suitable cationic lipids for use in the compositions andmethods of the invention include those described in international patentpublications WO 2010/053572 (and particularly, C12-200 described atparagraph [00225]) and WO 2012/170930, both of which are incorporatedherein by reference. In certain embodiments, the compositions andmethods of the invention employ a lipid nanoparticles comprising anionizable cationic lipid described in U.S. provisional patentapplication 61/617,468, filed Mar. 29, 2012 (incorporated herein byreference), such as, e.g, (15Z, 18Z)—N,N-dimethyl-6-(9Z,12Z)-octadeca-9, 12-dien-1-yl)tetracosa-15,18-dien-1-amine (HGT5000),(15Z, 18Z)—N,N-dimethyl-6-((9Z, 12Z)-octadeca-9,12-dien-1-yl)tetracosa-4,15,18-trien-1-amine (HGT5001), and(15Z,18Z)—N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl)tetracosa-5,15,18-trien-1-amine (HGT5002).

In some embodiments, the second or additional cationic lipidN-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride or “DOTMA”is used. (Feigner et al. (Proc. Nat'l Acad. Sci. 84, 7413 (1987); U.S.Pat. No. 4,897,355). DOTMA can be formulated alone or can be combinedwith the neutral lipid, dioleoylphosphatidyl-ethanolamine or “DOPE” orother cationic or non-cationic lipids into a liposomal transfer vehicleor a lipid nanoparticle, and such liposomes can be used to enhance thedelivery of nucleic acids into target cells. Other suitable cationiclipids include, for example, 5-carboxyspermylglycinedioctadecylamide or“DOGS,”2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminiumor “DOSPA” (Behr et al. Proc. Nat.'l Acad. Sci. 86, 6982 (1989); U.S.Pat. No. 5,171,678; U.S. Pat. No. 5,334,761),1,2-Dioleoyl-3-Dimethylammonium-Propane or “DODAP”,1,2-Dioleoyl-3-Trimethylammonium-Propane or “DOTAP”. Additionalexemplary cationic lipids also include1,2-distearyloxy-N,N-dimethyl-3-aminopropane or “DSDMA”,1,2-dioleyloxy-N,N-dimethyl-3-aminopropane or “DODMA”,1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or “DLinDMA”,1,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane or “DLenDMA”,N-dioleyl-N,N-dimethylammonium chloride or “DODAC”,N,N-distearyl-N,N-dimethylammonium bromide or “DDAB”,N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide or “DMRIE”,3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propaneor “CLinDMA”,2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,1-2′-octadecadienoxy)propaneor “CpLinDMA”, N,N-dimethyl-3,4-dioleyloxybenzylamine or “DMOBA”,1,2-N,N′-dioleylcarbamyl-3-dimethylaminopropane or “DOcarbDAP”,2,3-Dilinoleoyloxy-N,N-dimethylpropylamine or “DLinDAP”,1,2-N,N′-Dilinoleylcarbamyl-3-dimethylaminopropane or “DLincarbDAP”,1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane or “DLinCDAP”,2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane or “DLin-DMA”,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane or“DLin-K-XTC2-DMA”, and2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)-1,3-dioxolan-4-yl)-N,N-dimethylethanamine(DLin-KC2-DMA)) (See, WO 2010/042877; Semple et al., Nature Biotech. 28:172-176 (2010)), or mixtures thereof. (Heyes, J., et al., J ControlledRelease 107: 276-287 (2005); Morrissey, D V., et al., Nat. Biotechnol.23(8): 1003-1007 (2005); PCT Publication WO2005/121348A1). In someembodiments, one or more of the cationic lipids comprise at least one ofan imidazole, dialkylamino, or guanidinium moiety.

In some embodiments, the second or additional cationic lipid may bechosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane),MC3 (((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate), ALNY-100 ((3 aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)),NC98-5(4,7,13-tris(3-oxo-3-(undecylamino)propyl)-N1,N16-diundecyl-4,7,10,13-tetraazahexadecane-1,16-diamide),DODAP (1,2-dioleyl-3-dimethylammonium propane), HGT4003 (WO 2012/170889,the teachings of which are incorporated herein by reference in theirentirety), ICE (WO 2011/068810, the teachings of which are incorporatedherein by reference in their entirety), HGT5000 (U.S. Provisional PatentApplication No. 61/617,468, the teachings of which are incorporatedherein by reference in their entirety) or HGT5001 (cis or trans)(Provisional Patent Application No. 61/617,468), aminoalcohol lipidoidssuch as those disclosed in WO2010/053572, DOTAP(1,2-dioleyl-3-trimethylammonium propane), DOTMA(1,2-di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (Heyes, J.;Palmer, L.; Bremner, K.; MacLachlan, I. “Cationic lipid saturationinfluences intracellular delivery of encapsulated nucleic acids” J.Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (Semple, S. C. et al.“Rational Design of Cationic Lipids for siRNA Delivery” Nature Biotech.2010, 28, 172-176), C12-200 (Love, K. T. et al. “Lipid-like materialsfor low-dose in vivo gene silencing” PNAS 2010, 107, 1864-1869).

Non-Cationic/Helper Lipids

In some embodiments, provided liposomes contain one or more non-cationic(“helper”) lipids. As used herein, the phrase “non-cationic lipid”refers to any neutral, zwitterionic or anionic lipid. As used herein,the phrase “anionic lipid” refers to any of a number of lipid speciesthat carry a net negative charge at a selected H, such as physiologicalpH. Non-cationic lipids include, but are not limited to,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoylphosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixturethereof.

In some embodiments, such non-cationic lipids may be used alone, but arepreferably used in combination with other excipients, for example,cationic lipids. In some embodiments, the non-cationic lipid maycomprise a molar ratio of about 5% to about 90%, or about 10% to about70% of the total lipid present in a liposome. In some embodiments, anon-cationic lipid is a neutral lipid, i.e., a lipid that does not carrya net charge in the conditions under which the composition is formulatedand/or administered. In some embodiments, the percentage of non-cationiclipid in a liposome may be greater than 5%, greater than 10%, greaterthan 20%, greater than 30%, or greater than 40%.

Cholesterol-Based Lipids

In some embodiments, provided liposomes comprise one or morecholesterol-based lipids. For example, suitable cholesterol-basedcationic lipids include, for example, DC-Choi(N,N-dimethyl-N-ethylcarboxamidocholesterol),1,4-bis(3-N-oleylamino-propyl)piperazine (Gao, et al. Biochem. Biophys.Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997);U.S. Pat. No. 5,744,335), or ICE. In some embodiments, thecholesterol-based lipid may comprise a molar ration of about 2% to about30%, or about 5% to about 20% of the total lipid present in a liposome.In some embodiments, The percentage of cholesterol-based lipid in thelipid nanoparticle may be greater than 5, %, 10%, greater than 20%,greater than 30%, or greater than 40%.

PEGylated Lipids

In some embodiments, provided liposomes comprise one or more PEGylatedlipids. For example, the use of polyethylene glycol (PEG)-modifiedphospholipids and derivatized lipids such as derivatized ceramides(PEG-CER), including N-Octanoyl-Sphingosine-1-[Succinyl(MethoxyPolyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplatedby the present invention in combination with one or more of the cationicand, in some embodiments, other lipids together which comprise theliposome. Contemplated PEG-modified lipids include, but are not limitedto, a polyethylene glycol chain of up to 5 kDa in length covalentlyattached to a lipid with alkyl chain(s) of C₆-C₂₀ length. In someembodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterolor PEG-2K. The addition of such components may prevent complexaggregation and may also provide a means for increasing circulationlifetime and increasing the delivery of the lipid-nucleic acidcomposition to the target cell, (Klibanov et al. (1990) FEBS Letters,268 (1): 235-237), or they may be selected to rapidly exchange out ofthe formulation in vivo (see U.S. Pat. No. 5,885,613).

In some embodiments, particularly useful exchangeable lipids arePEG-ceramides having shorter acyl chains (e.g., C₁₄ or C₁₈). ThePEG-modified phospholipid and derivitized lipids of the presentinvention may comprise a molar ratio from about 0% to about 15%, about0.5% to about 15%, about 1% to about 15%, about 4% to about 10%, orabout 2% of the total lipid present in the liposome.

According to various embodiments, the selection of second or additionalcationic lipids, non-cationic lipids and/or PEG-modified lipids whichcomprise the lipid nanoparticle, as well as the relative molar ratio ofsuch lipids to each other, is based upon the characteristics of theselected lipid(s), the nature of the intended target cells, thecharacteristics of the mRNA to be delivered. Additional considerationsinclude, for example, the saturation of the alkyl chain, as well as thesize, charge, pH, pKa, fusogenicity and toxicity of the selectedlipid(s). Thus the molar ratios may be adjusted accordingly. In someembodiments, the percentage of PEG-modified lipid in a liposome may begreater than 1%, greater than 2%, greater than 5%, greater than 10%, orgreater than 15%.

Polymer

In some embodiments, a suitable liposome according to the presentinvention further includes a polymer, in combination with one or morecationic lipids as described and, in some embodiments, other carriersincluding various lipids described herein. Thus, in some embodiments,liposomal delivery vehicles, as used herein, also encompass polymercontaining nanoparticles. Suitable polymers may include, for example,polyacrylates, polyalkycyanoacrylates, polylactide,polylactide-polyglycolide copolymers, polycaprolactones, dextran,albumin, gelatin, alginate, collagen, chitosan, cyclodextrins,protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine(PEI). When PEI is present, it may be branched PEI of a molecular weightranging from 10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).

In some embodiments, a suitable liposome formulation contains acombination of one or more cationic lipids, one or more non-cationiclipids, one or more cholesterol-based lipids one or more PEG-modifiedlipids, and/or one or more polymers. As a non-limiting example, asuitable liposome comprises cKK-E12, DOPE, cholesterol and DMG-PEG2K. Insome embodiments, the ratio of cationic lipid to non-cationic lipid tocholesterol-based lipid to PEGylated lipid may be between about30-50:25-35:20-30:1-15, respectively. In some embodiments, the ratio ofcationic lipid to non-cationic lipid to cholesterol-based lipid toPEGylated lipid is approximately 40:30:20:10, respectively. In someembodiments, the ratio of cationic lipid to non-cationic lipid tocholesterol-based lipid to PEGylated lipid is approximately 40:30:25:5,respectively. In some embodiments, the ratio of cationic lipid tonon-cationic lipid to cholesterol-based lipid to PEGylated lipid isapproximately 40:32:25:3, respectively.

mRNA

The present invention can be used to deliver any mRNA. mRNA is typicallythought of as the type of RNA that carries information from DNA to theribosome. The existence of mRNA is usually very brief and includesprocessing and translation, followed by degradation. Typically, ineukaryotic organisms, mRNA processing comprises the addition of a “cap”on the N-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Atypical cap is a 7-methylguanosine cap, which is a guanosine that islinked through a 5′-5′-triphosphate bond to the first transcribednucleotide. The presence of the cap is important in providing resistanceto nucleases found in most eukaryotic cells. The tail is typically apolyadenylation event whereby a polyadenylyl moiety is added to the 3′end of the mRNA molecule. The presence of this “tail” serves to protectthe mRNA from exonuclease degradation. Messenger RNA typically istranslated by the ribosomes into a series of amino acids that make up aprotein.

Any mRNA capable of being translated into one or more peptides (e.g.,proteins) or peptide fragments is contemplated as within the scope ofthe present invention. In some embodiments, an mRNA encodes one or morenaturally occurring peptides. In some embodiments, an mRNA encodes oneor more modified or non-natural peptides.

In some embodiments an mRNA encodes an intracellular protein. In someembodiments, an mRNA encodes a cytosolic protein. In some embodiments,an mRNA encodes a protein associated with the actin cytoskeleton. Insome embodiments, an mRNA encodes a protein associated with the plasmamembrane. In some specific embodiments, an mRNA encodes a transmembraneprotein. In some specific embodiments an mRNA encodes an ion channelprotein. In some embodiments, an mRNA encodes a perinuclear protein. Insome embodiments, an mRNA encodes a nuclear protein. In some specificembodiments, an mRNA encodes a transcription factor. In someembodiments, an mRNA encodes a chaperone protein. In some embodiments,an mRNA encodes an intracellular enzyme (e.g., mRNA encoding an enzymeassociated with urea cycle or lysosomal storage metabolic disorders). Insome embodiments, an mRNA encodes a protein involved in cellularmetabolism, DNA repair, transcription and/or translation. In someembodiments, an mRNA encodes an extracellular protein. In someembodiments, an mRNA encodes a protein associated with the extracellularmatrix. In some embodiments an mRNA encodes a secreted protein. Inspecific embodiments, an mRNA used in the composition and methods of theinvention may be used to express functional proteins or enzymes that areexcreted or secreted by one or more target cells into the surroundingextracellular fluid (e.g., mRNA encoding hormones and/orneurotransmitters).

In some embodiments, the compositions and methods of the inventionprovide for delivery of mRNA encoding a secreted protein. In someembodiments, the compositions and methods of the invention provide fordelivery of mRNA encoding one or more secreted proteins listed in Table1; thus, compositions of the invention may comprise an mRNA encoding aprotein listed in Table 1 (or a homolog thereof) along with othercomponents set out herein, and methods of the invention may comprisepreparing and/or administering a composition comprising an mRNA encodinga protein listed in Table 1 (or a homolog thereof) along with othercomponents set out herein.

TABLE 1 Secreted Proteins Uniprot ID Protein Name Gene Name A1E959Odontogenic ameloblast-associated protein ODAM A1KZ92 Peroxidasin-likeprotein PXDNL A1L453 Serine protease 38 PRSS38 A1L4H1 Soluble scavengerreceptor cysteine-rich domain- SSC5D containing protein SSC5D A2RUU4Colipase-like protein 1 CLPSL1 A2VDF0 Fucose mutarotase FUOM A2VEC9SCO-spondin SSPO A3KMH1 von Willebrand factor A domain-containingprotein 8 VWA8 A4D0S4 Laminin subunit beta-4 LAMB4 A4D1T9 Probableinactive serine protease 37 PRSS37 A5D8T8 C-type lectin domain family 18member A CLEC18A A6NC86 phospholipase A2 inhibitor and Ly6/PLAUR PINLYPdomain-containing protein A6NCI4 von Willebrand factor Adomain-containing protein 3A VWA3A A6ND01 Probable folate receptor deltaFOLR4 A6NDD2 Beta-defensin 108B-like A6NE02 BTB/POZ domain-containingprotein 17 BTBD17 A6NEF6 Growth hormone 1 GH1 A6NF02 NPIP-like proteinLOC730153 A6NFB4 HCG1749481, isoform CRA_k CSH1 A6NFZ4 Protein FAM24AFAM24A A6NG13 Glycosyltransferase 54 domain-containing protein A6NGN9IgLON family member 5 IGLON5 A6NHN0 Otolin-1 OTOL1 A6NHN6 Nuclear porecomplex-interacting protein-like 2 NPIPL2 A6NI73 Leukocyteimmunoglobulin-like receptor LILRA5 subfamily A member 5 A6NIT4Chorionic somatomammotropin hormone 2 CSH2 isoform 2 A6NJ69 IgA-inducingprotein homolog IGIP A6NKQ9 Choriogonadotropin subunit beta variant 1CGB1 A6NMZ7 Collagen alpha-6(VI) chain COL6A6 A6NNS2Dehydrogenase/reductase SDR family member 7C DHRS7C A6XGL2 Insulin Achain INS A8K0G1 Protein Wnt WNT7B A8K2U0 Alpha-2-macroglobulin-likeprotein 1 A2ML1 A8K7I4 Calcium-activated chloride channel regulator 1CLCA1 A8MTL9 Serpin-like protein HMSD HMSD A8MV23 Serpin E3 SERPINE3A8MZH6 Oocyte-secreted protein 1 homolog OOSP1 A8TX70 Collagenalpha-5(VI) chain COL6A5 B0ZBE8 Natriuretic peptide NPPA B1A4G9Somatotropin GH1 B1A4H2 HCG1749481, isoform CRA_d CSH1 B1A4H9 Chorionicsomatomammotropin hormone CSH2 B1AJZ6 Protein Wnt WNT4 B1AKI9 Isthmin-1ISM1 B2RNN3 Complement C1q and tumor necrosis factor- C1QTNF9B relatedprotein 9B B2RUY7 von Willebrand factor C domain-containing VWC2Lprotein 2-like B3GLJ2 Prostate and testis expressed protein 3 PATE3B4DI03 SEC11-like 3 (S. cerevisiae), isoform CRA_a SEC11L3 B4DJF9Protein Wnt WNT4 B4DUL4 SEC11-like 1 (S. cerevisiae), isoform CRA_dSEC11L1 B5MCC8 Protein Wnt WNT10B B8A595 Protein Wnt WNT7B B8A597Protein Wnt WNT7B B8A598 Protein Wnt WNT7B B9A064 Immunoglobulinlambda-like polypeptide 5 IGLL5 C9J3H3 Protein Wnt WNT10B C9J8I8 ProteinWnt WNT5A C9JAF2 Insulin-like growth factor II Ala-25 Del IGF2 C9JCI2Protein Wnt WNT10B C9JL84 HERV-H LTR-associating protein 1 HHLA1 C9JNR5Insulin A chain INS C9JUI2 Protein Wnt WNT2 D6RF47 Protein Wnt WNT8AD6RF94 Protein Wnt WNT8A E2RYF7 Protein PBMUCL2 HCG22 E5RFR1PENK(114-133) PENK E7EML9 Serine protease 44 PRSS44 E7EPC3 Protein WntWNT9B E7EVP0 Nociceptin PNOC E9PD02 Insulin-like growth factor I IGF1E9PH60 Protein Wnt WNT16 E9PJL6 Protein Wnt WNT11 F5GYM2 Protein WntWNT5B F5H034 Protein Wnt WNT5B F5H364 Protein Wnt WNT5B F5H7Q6 ProteinWnt WNT5B F8WCM5 Protein INS-IGF2 INS-IGF2 F8WDR1 Protein Wnt WNT2H0Y663 Protein Wnt WNT4 H0YK72 Signal peptidase complex catalyticsubunit SEC11A SEC11A H0YK83 Signal peptidase complex catalytic subunitSEC11A SEC11A H0YM39 Chorionic somatomammotropin hormone CSH2 H0YMT7Chorionic somatomammotropin hormone CSH1 H0YN17 Chorionicsomatomammotropin hormone CSH2 H0YNA5 Signal peptidase complex catalyticsubunit SEC11A SEC11A H0YNG3 Signal peptidase complex catalytic subunitSEC11A SEC11A H0YNX5 Signal peptidase complex catalytic subunit SEC11ASEC11A H7BZB8 Protein Wnt WNT10A H9KV56 Choriogonadotropin subunit betavariant 2 CGB2 I3L0L8 Protein Wnt WNT9B J3KNZ1 Choriogonadotropinsubunit beta variant 1 CGB1 J3KP00 Choriogonadotropin subunit beta CGB7J3QT02 Choriogonadotropin subunit beta variant 1 CGB1 O00175 C-C motifchemokine 24 CCL24 O00182 Galectin-9 LGALS9 O00187 Mannan-binding lectinserine protease 2 MASP2 O00230 Cortistatin CORT O00253 Agouti-relatedprotein AGRP O00270 12-(S)-hydroxy-5,8,10,14-eicosatetraenoic acid GPR31receptor O00292 Left-right determination factor 2 LEFTY2 O00294Tubby-related protein 1 TULP1 O00295 Tubby-related protein 2 TULP2O00300 Tumor necrosis factor receptor superfamily TNFRSF11B member 11BO00339 Matrilin-2 MATN2 O00391 Sulfhydryl oxidase 1 QSOX1 O00468 AgrinAGRN O00515 Ladinin-1 LAD1 O00533 Processed neural cell adhesionmolecule L1-like CHL1 protein O00584 Ribonuclease T2 RNASET2 O00585 C-Cmotif chemokine 21 CCL21 O00602 Ficolin-1 FCN1 O00622 Protein CYR61CYR61 O00626 MDC(5-69) CCL22 O00634 Netrin-3 NTN3 O00744 Protein Wnt-10bWNT10B O00755 Protein Wnt-7a WNT7A O14498 Immunoglobulin superfamilycontaining leucine- ISLR rich repeat protein O14511 Pro-neuregulin-2,membrane-bound isoform NRG2 O14594 Neurocan core protein NCAN O14625C—X—C motif chemokine 11 CXCL11 O14638 Ectonucleotide ENPP3pyrophosphatase/phosphodiesterase family member 3 O14656 Torsin-1A TOR1AO14657 Torsin-1B TOR1B O14786 Neuropilin-1 NRP1 O14788 Tumor necrosisfactor ligand superfamily member TNFSF11 11, membrane form O14791Apolipoprotein L1 APOL1 O14793 Growth/differentiation factor 8 MSTNO14904 Protein Wnt-9a WNT9A O14905 Protein Wnt-9b WNT9B O14944Proepiregulin EREG O14960 Leukocyte cell-derived chemotaxin-2 LECT2O15018 Processed PDZ domain-containing protein 2 PDZD2 O15041Semaphorin-3E SEMA3E O15072 A disintegrin and metalloproteinase withADAMTS3 thrombospondin motifs 3 O15123 Angiopoietin-2 ANGPT2 O15130Neuropeptide FF NPFF O15197 Ephrin type-B receptor 6 EPHB6 O15204ADAMDEC1 ADAMDEC1 O15230 Laminin subunit alpha-5 LAMA5 O15232 Matrilin-3MATN3 O15240 Neuroendocrine regulatory peptide-1 VGF O15263Beta-defensin 4A DEFB4A O15335 Chondroadherin CHAD O15393 Transmembraneprotease serine 2 catalytic chain TMPRSS2 O15444 C-C motif chemokine 25CCL25 O15467 C-C motif chemokine 16 CCL16 O15496 Group 10 secretoryphospholipase A2 PLA2G10 O15520 Fibroblast growth factor 10 FGF10 O15537Retinoschisin RS1 O43157 Plexin-B1 PLXNB1 O43184 Disintegrin andmetalloproteinase domain- ADAM12 containing protein 12 O43240Kallikrein-10 KLK10 O43278 Kunitz-type protease inhibitor 1 SPINT1O43320 Fibroblast growth factor 16 FGF16 O43323 Desert hedgehog proteinC-product DHH O43405 Cochlin COCH O43508 Tumor necrosis factor ligandsuperfamily member TNFSF12 12, membrane form O43555 Progonadoliberin-2GNRH2 O43557 Tumor necrosis factor ligand superfamily member TNFSF14 14,soluble form O43692 Peptidase inhibitor 15 PI15 O43699 Sialicacid-binding Ig-like lectin 6 SIGLEC6 O43820 Hyaluronidase-3 HYAL3O43827 Angiopoietin-related protein 7 ANGPTL7 O43852 Calumenin CALUO43854 EGF-like repeat and discoidin I-like domain- EDIL3 containingprotein 3 O43866 CD5 antigen-like CD5L O43897 Tolloid-like protein 1TLL1 O43915 Vascular endothelial growth factor D FIGF O43927 C—X—C motifchemokine 13 CXCL13 O60218 Aldo-keto reductase family 1 member B10AKR1B10 O60235 Transmembrane protease serine 11D TMPRSS11D O60258Fibroblast growth factor 17 FGF17 O60259 Kallikrein-8 KLK8 O60383Growth/differentiation factor 9 GDF9 O60469 Down syndrome cell adhesionmolecule DSCAM O60542 Persephin PSPN O60565 Gremlin-1 GREM1 O60575Serine protease inhibitor Kazal-type 4 SPINK4 O60676 Cystatin-8 CST8O60687 Sushi repeat-containing protein SRPX2 SRPX2 O60844 Zymogengranule membrane protein 16 ZG16 O60882 Matrix metalloproteinase-20MMP20 O60938 Keratocan KERA O75015 Low affinity immunoglobulin gamma Fcregion FCGR3B receptor III-B O75077 Disintegrin and metalloproteinasedomain- ADAM23 containing protein 23 O75093 Slit homolog 1 protein SLIT1O75094 Slit homolog 3 protein SLIT3 O75095 Multiple epidermal growthfactor-like domains MEGF6 protein 6 O75173 A disintegrin andmetalloproteinase with ADAMTS4 thrombospondin motifs 4 O75200 Nuclearpore complex-interacting protein-like 1 NPIPL1 O75339 Cartilageintermediate layer protein 1 C1 CILP O75354 Ectonucleoside triphosphatediphosphohydrolase 6 ENTPD6 O75386 Tubby-related protein 3 TULP3 O75398Deformed epidermal autoregulatory factor 1 DEAF1 homolog O75443Alpha-tectorin TECTA O75445 Usherin USH2A O75462 Cytokine receptor-likefactor 1 CRLF1 O75487 Glypican-4 GPC4 O75493 Carbonic anhydrase-relatedprotein 11 CA11 O75594 Peptidoglycan recognition protein 1 PGLYRP1O75596 C-type lectin domain family 3 member A CLEC3A O75610 Left-rightdetermination factor 1 LEFTY1 O75629 Protein CREG1 CREG1 O75636Ficolin-3 FCN3 O75711 Scrapie-responsive protein 1 SCRG1 O75715Epididymal secretory glutathione peroxidase GPX5 O75718Cartilage-associated protein CRTAP O75829 Chondrosurfactant proteinLECT1 O75830 Serpin I2 SERPINI2 O75882 Attractin ATRN O75888 Tumornecrosis factor ligand superfamily member TNFSF13 13 O75900 Matrixmetalloproteinase-23 MMP23A O75951 Lysozyme-like protein 6 LYZL6 O75973C1q-related factor C1QL1 O76038 Secretagogin SCGN O76061 Stanniocalcin-2STC2 O76076 WNT1-inducible-signaling pathway protein 2 WISP2 O76093Fibroblast growth factor 18 FGF18 O76096 Cystatin-F CST7 O94769Extracellular matrix protein 2 ECM2 O94813 Slit homolog 2 proteinC-product SLIT2 O94907 Dickkopf-related protein 1 DKK1 O94919Endonuclease domain-containing 1 protein ENDOD1 O94964 N-terminal formSOGA1 O95025 Semaphorin-3D SEMA3D O95084 Serine protease 23 PRSS23O95150 Tumor necrosis factor ligand superfamily member TNFSF15 15 O95156Neurexophilin-2 NXPH2 O95157 Neurexophilin-3 NXPH3 O95158Neurexophilin-4 NXPH4 O95388 WNT1-inducible-signaling pathway protein 1WISP1 O95389 WNT1-inducible-signaling pathway protein 3 WISP3 O95390Growth/differentiation factor 11 GDF11 O95393 Bone morphogenetic protein10 BMP10 O95399 Urotensin-2 UTS2 O95407 Tumor necrosis factor receptorsuperfamily TNFRSF6B member 6B O95428 Papilin PAPLN O95445Apolipoprotein M APOM O95450 A disintegrin and metalloproteinase withADAMTS2 thrombospondin motifs 2 O95460 Matrilin-4 MATN4 O95467 LHALtetrapeptide GNAS O95631 Netrin-1 NTN1 O95633 Follistatin-relatedprotein 3 FSTL3 O95711 Lymphocyte antigen 86 LY86 O95715 C—X—C motifchemokine 14 CXCL14 O95750 Fibroblast growth factor 19 FGF19 O95760Interleukin-33 IL33 O95813 Cerberus CER1 O95841 Angiopoietin-relatedprotein 1 ANGPTL1 O95897 Noelin-2 OLFM2 O95925 Eppin EPPIN O95965Integrin beta-like protein 1 ITGBL1 O95967 EGF-containing fibulin-likeextracellular matrix EFEMP2 protein 2 O95968 Secretoglobin family 1Dmember 1 SCGB1D1 O95969 Secretoglobin family 1D member 2 SCGB1D2 O95970Leucine-rich glioma-inactivated protein 1 LGI1 O95972 Bone morphogeneticprotein 15 BMP15 O95994 Anterior gradient protein 2 homolog AGR2 O95998Interleukin-18-binding protein IL18BP O96009 Napsin-A NAPSA O96014Protein Wnt-11 WNT11 P00450 Ceruloplasmin CP P00451 Factor VIIIa lightchain F8 P00488 Coagulation factor XIII A chain F13A1 P00533 Epidermalgrowth factor receptor EGFR P00709 Alpha-lactalbumin LALBA P00734Prothrombin F2 P00738 Haptoglobin beta chain HP P00739Haptoglobin-related protein HPR P00740 Coagulation factor IXa heavychain F9 P00742 Factor X heavy chain F10 P00746 Complement factor D CFDP00747 Plasmin light chain B PLG P00748 Coagulation factor XIIa lightchain F12 P00749 Urokinase-type plasminogen activator long chain A PLAUP00750 Tissue-type plasminogen activator PLAT P00751 Complement factor BBa fragment CFB P00797 Renin REN P00973 2′-5′-oligoadenylate synthase 1OAS1 P00995 Pancreatic secretory trypsin inhibitor SPINK1 P01008Antithrombin-III SERPINC1 P01009 Alpha-1-antitrypsin SERPINA1 P01011Alpha-1-antichymotrypsin His-Pro-less SERPINA3 P01019 Angiotensin-1 AGTP01023 Alpha-2-macroglobulin A2M P01024 Acylation stimulating protein C3P01031 Complement C5 beta chain C5 P01033 Metalloproteinase inhibitor 1TIMP1 P01034 Cystatin-C CST3 P01036 Cystatin-S CST4 P01037 Cystatin-SNCST1 P01042 Kininogen-1 light chain KNG1 P01127 Platelet-derived growthfactor subunit B PDGFB P01135 Transforming growth factor alpha TGFAP01137 Transforming growth factor beta-1 TGFB1 P01138 Beta-nerve growthfactor NGF P01148 Gonadoliberin-1 GNRH1 P01160 Atrial natriuretic factorNPPA P01178 Oxytocin OXT P01185 Vasopressin-neurophysin 2-copeptin AVPP01189 Corticotropin POMC P01210 PENK(237-258) PENK P01213Alpha-neoendorphin PDYN P01215 Glycoprotein hormones alpha chain CGAP01222 Thyrotropin subunit beta TSHB P01225 Follitropin subunit betaFSHB P01229 Lutropin subunit beta LHB P01233 Choriogonadotropin subunitbeta CGB8 P01236 Prolactin PRL P01241 Somatotropin GH1 P01242 Growthhormone variant GH2 P01243 Chorionic somatomammotropin hormone CSH2P01258 Katacalcin CALCA P01266 Thyroglobulin TG P01270 Parathyroidhormone PTH P01275 Glucagon GCG P01282 Intestinal peptide PHM-27 VIPP01286 Somatoliberin GHRH P01298 Pancreatic prohormone PPY P01303C-flanking peptide of NPY NPY P01308 Insulin INS P01344 Insulin-likegrowth factor II IGF2 P01350 Big gastrin GAST P01374 Lymphotoxin-alphaLTA P01375 C-domain 1 TNF P01562 Interferon alpha-1/13 IFNA1 P01563Interferon alpha-2 IFNA2 P01566 Interferon alpha-10 IFNA10 P01567Interferon alpha-7 IFNA7 P01568 Interferon alpha-21 IFNA21 P01569Interferon alpha-5 IFNA5 P01570 Interferon alpha-14 IFNA14 P01571Interferon alpha-17 IFNA17 P01574 Interferon beta IFNB1 P01579Interferon gamma IFNG P01583 Interleukin-1 alpha IL1A P01584Interleukin-1 beta IL1B P01588 Erythropoietin EPO P01591 ImmunoglobulinJ chain IGJ P01732 T-cell surface glycoprotein CD8 alpha chain CD8AP01833 Polymeric immunoglobulin receptor PIGR P01857 Ig gamma-1 chain Cregion IGHG1 P01859 Ig gamma-2 chain C region IGHG2 P01860 Ig gamma-3chain C region IGHG3 P01861 Ig gamma-4 chain C region IGHG4 P01871 Ig muchain C region IGHM P01880 Ig delta chain C region IGHD P02452 Collagenalpha-1(I) chain COL1A1 P02458 Chondrocalcin COL2A1 P02461 Collagenalpha-1(III) chain COL3A1 P02462 Collagen alpha-1(IV) chain COL4A1P02647 Apolipoprotein A-I APOA1 P02649 Apolipoprotein E APOE P02652Apolipoprotein A-II APOA2 P02654 Apolipoprotein C-I APOC1 P02655Apolipoprotein C-II APOC2 P02656 Apolipoprotein C-III APOC3 P02671Fibrinogen alpha chain FGA P02675 Fibrinopeptide B FGB P02679 Fibrinogengamma chain FGG P02741 C-reactive protein CRP P02743 Serum amyloidP-component(1-203) APCS P02745 Complement C1q subcomponent subunit AC1QA P02746 Complement C1q subcomponent subunit B C1QB P02747 ComplementC1q subcomponent subunit C C1QC P02748 Complement component C9b C9P02749 Beta-2-glycoprotein 1 APOH P02750 Leucine-richalpha-2-glycoprotein LRG1 P02751 Ugl-Y2 FN1 P02753 Retinol-bindingprotein 4 RBP4 P02760 Trypstatin AMBP P02763 Alpha-1-acid glycoprotein 1ORM1 P02765 Alpha-2-HS-glycoprotein chain A AHSG P02766 TransthyretinTTR P02768 Serum albumin ALB P02771 Alpha-fetoprotein AFP P02774 VitaminD-binding protein GC P02775 Connective tissue-activating peptide IIIPPBP P02776 Platelet factor 4 PF4 P02778 CXCL10(1-73) CXCL10 P02786Transferrin receptor protein 1 TFRC P02787 Serotransferrin TF P02788Lactoferroxin-C LTF P02790 Hemopexin HPX P02808 Statherin STATH P02810Salivary acidic proline-rich phosphoprotein 1/2 PRH2 P02812 Basicsalivary proline-rich protein 2 PRB2 P02814 Peptide D1A SMR3B P02818Osteocalcin BGLAP P03950 Angiogenin ANG P03951 Coagulation factor XIaheavy chain F11 P03952 Plasma kallikrein KLKB1 P03956 27 kDainterstitial collagenase MMP1 P03971 Muellerian-inhibiting factor AMHP03973 Antileukoproteinase SLPI P04003 C4b-binding protein alpha chainC4BPA P04004 Somatomedin-B VTN P04054 Phospholipase A2 PLA2G1B P04085Platelet-derived growth factor subunit A PDGFA P04090 Relaxin A chainRLN2 P04114 Apolipoprotein B-100 APOB P04118 Colipase CLPS P04141Granulocyte-macrophage colony-stimulating CSF2 factor P04155 Trefoilfactor 1 TFF1 P04180 Phosphatidylcholine-sterol acyltransferase LCATP04196 Histidine-rich glycoprotein HRG P04217 Alpha-1B-glycoprotein A1BGP04275 von Willebrand antigen 2 VWF P04278 Sex hormone-binding globulinSHBG P04279 Alpha-inhibin-31 SEMG1 P04280 Basic salivary proline-richprotein 1 PRB1 P04628 Proto-oncogene Wnt-1 WNT1 P04745 Alpha-amylase 1AMY1A P04746 Pancreatic alpha-amylase AMY2A P04808 Prorelaxin H1 RLN1P05000 Interferon omega-1 IFNW1 P05013 Interferon alpha-6 IFNA6 P05014Interferon alpha-4 IFNA4 P05015 Interferon alpha-16 IFNA16 P05019Insulin-like growth factor I IGF1 P05060 GAWK peptide CHGB P05090Apolipoprotein D APOD P05109 Protein S100-A8 S100A8 P05111 Inhibin alphachain INHA P05112 Interleukin-4 IL4 P05113 Interleukin-5 IL5 P05120Plasminogen activator inhibitor 2 SERPINB2 P05121 Plasminogen activatorinhibitor 1 SERPINE1 P05154 Plasma serine protease inhibitor SERPINA5P05155 Plasma protease C1 inhibitor SERPING1 P05156 Complement factor Iheavy chain CFI P05160 Coagulation factor XIII B chain F13B P05161Ubiquitin-like protein ISG15 ISG15 P05230 Fibroblast growth factor 1FGF1 P05231 Interleukin-6 IL6 P05305 Big endothelin-1 EDN1 P05408C-terminal peptide SCG5 P05451 Lithostathine-1-alpha REG1A P05452Tetranectin CLEC3B P05543 Thyroxine-binding globulin SERPINA7 P05814Beta-casein CSN2 P05997 Collagen alpha-2(V) chain COL5A2 P06276Cholinesterase BCHE P06307 Cholecystokinin-12 CCK P06396 Gelsolin GSNP06681 Complement C2 C2 P06702 Protein S100-A9 S100A9 P06727Apolipoprotein A-IV APOA4 P06734 Low affinity immunoglobulin epsilon Fcreceptor FCER2 soluble form P06744 Glucose-6-phosphate isomerase GPIP06850 Corticoliberin CRH P06858 Lipoprotein lipase LPL P06881Calcitonin gene-related peptide 1 CALCA P07093 Glia-derived nexinSERPINE2 P07098 Gastric triacylglycerol lipase LIPF P07225 VitaminK-dependent protein S PROS1 P07237 Protein disulfide-isomerase P4HBP07288 Prostate-specific antigen KLK3 P07306 Asialoglycoprotein receptor1 ASGR1 P07355 Annexin A2 ANXA2 P07357 Complement component C8 alphachain C8A P07358 Complement component C8 beta chain C8B P07360Complement component C8 gamma chain C8G P07477 Alpha-trypsin chain 2PRSS1 P07478 Trypsin-2 PRSS2 P07492 Neuromedin-C GRP P07498 Kappa-caseinCSN3 P07585 Decorin DCN P07911 Uromodulin UMOD P07942 Laminin subunitbeta-1 LAMB1 P07988 Pulmonary surfactant-associated protein B SFTPBP07998 Ribonuclease pancreatic RNASE1 P08118 Beta-microseminoproteinMSMB P08123 Collagen alpha-2(I) chain COL1A2 P08185Corticosteroid-binding globulin SERPINA6 P08217 Chymotrypsin-likeelastase family member 2A CELA2A P08218 Chymotrypsin-like elastasefamily member 2B CELA2B P08253 72 kDa type IV collagenase MMP2 P08254Stromelysin-1 MMP3 P08294 Extracellular superoxide dismutase [Cu—Zn]SOD3 P08476 Inhibin beta A chain INHBA P08493 Matrix Gla protein MGPP08572 Collagen alpha-2(IV) chain COL4A2 P08581 Hepatocyte growth factorreceptor MET P08603 Complement factor H CFH P08620 Fibroblast growthfactor 4 FGF4 P08637 Low affinity immunoglobulin gamma Fc region FCGR3Areceptor III-A P08697 Alpha-2-antiplasmin SERPINF2 P08700 Interleukin-3IL3 P08709 Coagulation factor VII F7 P08833 Insulin-like growthfactor-binding protein 1 IGFBP1 P08887 Interleukin-6 receptor subunitalpha IL6R P08949 Neuromedin-B-32 NMB P08F94 Fibrocystin PKHD1 P09038Fibroblast growth factor 2 FGF2 P09228 Cystatin-SA CST2 P09237Matrilysin MMP7 P09238 Stromelysin-2 MMP10 P09341 Growth-regulated alphaprotein CXCL1 P09382 Galectin-1 LGALS1 P09466 Glycodelin PAEP P09486SPARC SPARC P09529 Inhibin beta B chain INHBB P09544 Protein Wnt-2 WNT2P09603 Processed macrophage colony-stimulating factor 1 CSF1 P09681Gastric inhibitory polypeptide GIP P09683 Secretin SCT P09919Granulocyte colony-stimulating factor CSF3 P0C091 FRAS1-relatedextracellular matrix protein 3 FREM3 P0C0L4 C4d-A C4A P0C0L5 ComplementC4-B alpha chain C4B P0C0P6 Neuropeptide S NPS P0C7L1 Serine proteaseinhibitor Kazal-type 8 SPINK8 P0C862 Complement C1q and tumor necrosisfactor- C1QTNF9 related protein 9A P0C8F1 Prostate and testis expressedprotein 4 PATE4 P0CG01 Gastrokine-3 GKN3P P0CG36 Cryptic family protein1B CFC1B P0CG37 Cryptic protein CFC1 P0CJ68 Humanin-like protein 1MTRNR2L1 P0CJ69 Humanin-like protein 2 MTRNR2L2 P0CJ70 Humanin-likeprotein 3 MTRNR2L3 P0CJ71 Humanin-like protein 4 MTRNR2L4 P0CJ72Humanin-like protein 5 MTRNR2L5 P0CJ73 Humanin-like protein 6 MTRNR2L6P0CJ74 Humanin-like protein 7 MTRNR2L7 P0CJ75 Humanin-like protein 8MTRNR2L8 P0CJ76 Humanin-like protein 9 MTRNR2L9 P0CJ77 Humanin-likeprotein 10 MTRNR2L10 P0DJD7 Pepsin A-4 PGA4 P0DJD8 Pepsin A-3 PGA3P0DJD9 Pepsin A-5 PGA5 P0DJI8 Amyloid protein A SAA1 P0DJI9 Serumamyloid A-2 protein SAA2 P10082 Peptide YY(3-36) PYY P10092 Calcitoningene-related peptide 2 CALCB P10124 Serglycin SRGN P10145 MDNCF-a IL8P10147 MIP-1-alpha(4-69) CCL3 P10163 Peptide P-D PRB4 P10451 OsteopontinSPP1 P10599 Thioredoxin TXN P10600 Transforming growth factor beta-3TGFB3 P10643 Complement component C7 C7 P10645 Vasostatin-2 CHGA P10646Tissue factor pathway inhibitor TFPI P10720 Platelet factor 4variant(4-74) PF4V1 P10745 Retinol-binding protein 3 RBP3 P10767Fibroblast growth factor 6 FGF6 P10909 Clusterin alpha chain CLU P10912Growth hormone receptor GHR P10915 Hyaluronan and proteoglycan linkprotein 1 HAPLN1 P10966 T-cell surface glycoprotein CD8 beta chain CD8BP10997 Islet amyloid polypeptide IAPP P11047 Laminin subunit gamma-1LAMC1 P11150 Hepatic triacylglycerol lipase LIPC P11226 Mannose-bindingprotein C MBL2 P11464 Pregnancy-specific beta-1-glycoprotein 1 PSG1P11465 Pregnancy-specific beta-1-glycoprotein 2 PSG2 P11487 Fibroblastgrowth factor 3 FGF3 P11597 Cholesteryl ester transfer protein CETPP11684 Uteroglobin SCGB1A1 P11686 Pulmonary surfactant-associatedprotein C SFTPC P12034 Fibroblast growth factor 5 FGF5 P12107 Collagenalpha-1(XI) chain COL11A1 P12109 Collagen alpha-1(VI) chain COL6A1P12110 Collagen alpha-2(VI) chain COL6A2 P12111 Collagen alpha-3(VI)chain COL6A3 P12259 Coagulation factor V F5 P12272 PTHrP[1-36] PTHLHP12273 Prolactin-inducible protein PIP P12544 Granzyme A GZMA P12643Bone morphogenetic protein 2 BMP2 P12644 Bone morphogenetic protein 4BMP4 P12645 Bone morphogenetic protein 3 BMP3 P12724 Eosinophil cationicprotein RNASE3 P12821 Angiotensin-converting enzyme, soluble form ACEP12838 Neutrophil defensin 4 DEFA4 P12872 Motilin MLN P13232Interleukin-7 IL7 P13236 C-C motif chemokine 4 CCL4 P13284Gamma-interferon-inducible lysosomal thiol IFI30 reductase P13500 C-Cmotif chemokine 2 CCL2 P13501 C-C motif chemokine 5 CCL5 P13521Secretogranin-2 SCG2 P13591 Neural cell adhesion molecule 1 NCAM1 P13611Versican core protein VCAN P13671 Complement component C6 C6 P13688Carcinoembryonic antigen-related cell adhesion CEACAM1 molecule 1 P13725Oncostatin-M OSM P13726 Tissue factor F3 P13727 Eosinophil granule majorbasic protein PRG2 P13942 Collagen alpha-2(XI) chain COL11A2 P13987 CD59glycoprotein CD59 P14138 Endothelin-3 EDN3 P14174 Macrophage migrationinhibitory factor MIF P14207 Folate receptor beta FOLR2 P14222Perforin-1 PRF1 P14543 Nidogen-1 NID1 P14555 Phospholipase A2, membraneassociated PLA2G2A P14625 Endoplasmin HSP90B1 P14735 Insulin-degradingenzyme IDE P14778 Interleukin-1 receptor type 1, soluble form IL1R1P14780 82 kDa matrix metalloproteinase-9 MMP9 P15018 Leukemia inhibitoryfactor LIF P15085 Carboxypeptidase A1 CPA1 P15086 Carboxypeptidase BCPB1 P15151 Poliovirus receptor PVR P15169 Carboxypeptidase N catalyticchain CPN1 P15248 Interleukin-9 IL9 P15291 N-acetyllactosamine synthaseB4GALT1 P15309 PAPf39 ACPP P15328 Folate receptor alpha FOLR1 P15374Ubiquitin carboxyl-terminal hydrolase isozyme L3 UCHL3 P15502 ElastinELN P15509 Granulocyte-macrophage colony-stimulating CSF2RA factorreceptor subunit alpha P15515 Histatin-1 HTN1 P15516His3-(31-51)-peptide HTN3 P15692 Vascular endothelial growth factor AVEGFA P15814 Immunoglobulin lambda-like polypeptide 1 IGLL1 P15907Beta-galactoside alpha-2,6-sialyltransferase 1 ST6GAL1 P15941 Mucin-1subunit beta MUC1 P16035 Metalloproteinase inhibitor 2 TIMP2 P16112Aggrecan core protein 2 ACAN P16233 Pancreatic triacylglycerol lipasePNLIP P16442 Histo-blood group ABO system transferase ABO P16471Prolactin receptor PRLR P16562 Cysteine-rich secretory protein 2 CRISP2P16619 C-C motif chemokine 3-like 1 CCL3L1 P16860 BNP(3-29) NPPB P16870Carboxypeptidase E CPE P16871 Interleukin-7 receptor subunit alpha IL7RP17213 Bactericidal permeability-increasing protein BPI P17538Chymotrypsinogen B CTRB1 P17931 Galectin-3 LGALS3 P17936 Insulin-likegrowth factor-binding protein 3 IGFBP3 P17948 Vascular endothelialgrowth factor receptor 1 FLT1 P18065 Insulin-like growth factor-bindingprotein 2 IGFBP2 P18075 Bone morphogenetic protein 7 BMP7 P18428Lipopolysaccharide-binding protein LBP P18509 PACAP-related peptideADCYAP1 P18510 Interleukin-1 receptor antagonist protein IL1RN P18827Syndecan-1 SDC1 P19021 Peptidylglycine alpha-hydroxylating PAMmonooxygenase P19235 Erythropoietin receptor EPOR P19438 Tumor necrosisfactor-binding protein 1 TNFRSF1A P19652 Alpha-1-acid glycoprotein 2ORM2 P19801 Amiloride-sensitive amine oxidase [copper- ABP1 containing]P19823 Inter-alpha-trypsin inhibitor heavy chain H2 ITIH2 P19827Inter-alpha-trypsin inhibitor heavy chain H1 ITIH1 P19835 Bilesalt-activated lipase CEL P19875 C—X—C motif chemokine 2 CXCL2 P19876C—X—C motif chemokine 3 CXCL3 P19883 Follistatin FST P19957 Elafin PI3P19961 Alpha-amylase 2B AMY2B P20061 Transcobalamin-1 TCN1 P20062Transcobalamin-2 TCN2 P20142 Gastricsin PGC P20155 Serine proteaseinhibitor Kazal-type 2 SPINK2 P20231 Tryptase beta-2 TPSB2 P20333 Tumornecrosis factor receptor superfamily TNFRSF1B member 1B P20366 SubstanceP TAC1 P20382 Melanin-concentrating hormone PMCH P20396 Thyroliberin TRHP20742 Pregnancy zone protein PZP P20774 Mimecan OGN P20783Neurotrophin-3 NTF3 P20800 Endothelin-2 EDN2 P20809 Interleukin-11 IL11P20827 Ephrin-A1 EFNA1 P20849 Collagen alpha-1(IX) chain COL9A1 P20851C4b-binding protein beta chain C4BPB P20908 Collagen alpha-1(V) chainCOL5A1 P21128 Poly(U)-specific endoribonuclease ENDOU P21246Pleiotrophin PTN P21583 Kit ligand KITLG P21741 Midkine MDK P21754 Zonapellucida sperm-binding protein 3 ZP3 P21781 Fibroblast growth factor 7FGF7 P21802 Fibroblast growth factor receptor 2 FGFR2 P21810 BiglycanBGN P21815 Bone sialoprotein 2 IBSP P21860 Receptor tyrosine-proteinkinase erbB-3 ERBB3 P21941 Cartilage matrix protein MATN1 P22003 Bonemorphogenetic protein 5 BMP5 P22004 Bone morphogenetic protein 6 BMP6P22079 Lactoperoxidase LPO P22105 Tenascin-X TNXB P22301 Interleukin-10IL10 P22303 Acetylcholinesterase ACHE P22352 Glutathione peroxidase 3GPX3 P22362 C-C motif chemokine 1 CCL1 P22455 Fibroblast growth factorreceptor 4 FGFR4 P22466 Galanin message-associated peptide GAL P22692Insulin-like growth factor-binding protein 4 IGFBP4 P22749 GranulysinGNLY P22792 Carboxypeptidase N subunit 2 CPN2 P22891 Vitamin K-dependentprotein Z PROZ P22894 Neutrophil collagenase MMP8 P23142 Fibulin-1 FBLN1P23280 Carbonic anhydrase 6 CA6 P23352 Anosmin-1 KAL1 P23435Cerebellin-1 CBLN1 P23560 Brain-derived neurotrophic factor BDNF P23582C-type natriuretic peptide NPPC P23946 Chymase CMA1 P24043 Lamininsubunit alpha-2 LAMA2 P24071 Immunoglobulin alpha Fc receptor FCARP24347 Stromelysin-3 MMP11 P24387 Corticotropin-releasing factor-bindingprotein CRHBP P24592 Insulin-like growth factor-binding protein 6 IGFBP6P24593 Insulin-like growth factor-binding protein 5 IGFBP5 P24821Tenascin TNC P24855 Deoxyribonuclease-1 DNASE1 P25067 Collagenalpha-2(VIII) chain COL8A2 P25311 Zinc-alpha-2-glycoprotein AZGP1 P25391Laminin subunit alpha-1 LAMA1 P25445 Tumor necrosis factor receptorsuperfamily FAS member 6 P25940 Collagen alpha-3(V) chain COL5A3 P25942Tumor necrosis factor receptor superfamily CD40 member 5 P26022Pentraxin-related protein PTX3 PTX3 P26927 Hepatocyte growth factor-likeprotein beta chain MST1 P27169 Serum paraoxonase/arylesterase 1 PON1P27352 Gastric intrinsic factor GIF P27487 Dipeptidyl peptidase 4membrane form DPP4 P27539 Embryonic growth/differentiation factor 1 GDF1P27658 Vastatin COL8A1 P27797 Calreticulin CALR P27918 Properdin CFPP28039 Acyloxyacyl hydrolase AOAH P28300 Protein-lysine 6-oxidase LOXP28325 Cystatin-D CST5 P28799 Granulin-1 GRN P29122 Proproteinconvertase subtilisin/kexin type 6 PCSK6 P29279 Connective tissue growthfactor CTGF P29320 Ephrin type-A receptor 3 EPHA3 P29400 Collagenalpha-5(IV) chain COL4A5 P29459 Interleukin-12 subunit alpha IL12AP29460 Interleukin-12 subunit beta IL12B P29508 Serpin B3 SERPINB3P29622 Kallistatin SERPINA4 P29965 CD40 ligand, soluble form CD40LGP30990 Neurotensin/neuromedin N NTS P31025 Lipocalin-1 LCN1 P31151Protein S100-A7 S100A7 P31371 Fibroblast growth factor 9 FGF9 P31431Syndecan-4 SDC4 P31947 14-3-3 protein sigma SFN P32455Interferon-induced guanylate-binding protein 1 GBP1 P32881 Interferonalpha-8 IFNA8 P34096 Ribonuclease 4 RNASE4 P34130 Neurotrophin-4 NTF4P34820 Bone morphogenetic protein 8B BMP8B P35030 Trypsin-3 PRSS3 P35052Secreted glypican-1 GPC1 P35070 Betacellulin BTC P35225 Interleukin-13IL13 P35247 Pulmonary surfactant-associated protein D SFTPD P35318 ADMADM P35542 Serum amyloid A-4 protein SAA4 P35555 Fibrillin-1 FBN1 P35556Fibrillin-2 FBN2 P35625 Metalloproteinase inhibitor 3 TIMP3 P35858Insulin-like growth factor-binding protein complex IGFALS acid labilesubunit P35916 Vascular endothelial growth factor receptor 3 FLT4 P35968Vascular endothelial growth factor receptor 2 KDR P36222Chitinase-3-like protein 1 CHI3L1 P36952 Serpin B5 SERPINB5 P36955Pigment epithelium-derived factor SERPINF1 P36980 Complement factorH-related protein 2 CFHR2 P39059 Collagen alpha-1(XV) chain COL15A1P39060 Collagen alpha-1(XVIII) chain COL18A1 P39877 Calcium-dependentphospholipase A2 PLA2G5 P39900 Macrophage metalloelastase MMP12 P39905Glial cell line-derived neurotrophic factor GDNF P40225 ThrombopoietinTHPO P40967 M-alpha PMEL P41159 Leptin LEP P41221 Protein Wnt-5a WNT5AP41222 Prostaglandin-H2 D-isomerase PTGDS P41271 Neuroblastomasuppressor of tumorigenicity 1 NBL1 P41439 Folate receptor gamma FOLR3P42127 Agouti-signaling protein ASIP P42702 Leukemia inhibitory factorreceptor LIFR P42830 ENA-78(9-78) CXCL5 P43026 Growth/differentiationfactor 5 GDF5 P43251 Biotinidase BTD P43652 Afamin AFM P45452Collagenase 3 MMP13 P47710 Casoxin-D CSN1S1 P47929 Galectin-7 LGALS7BP47972 Neuronal pentraxin-2 NPTX2 P47989 Xanthine oxidase XDH P47992Lymphotactin XCL1 P48023 Tumor necrosis factor ligand superfamily memberFASLG 6, membrane form P48052 Carboxypeptidase A2 CPA2 P48061 Stromalcell-derived factor 1 CXCL12 P48304 Lithostathine-1-beta REG1B P48307Tissue factor pathway inhibitor 2 TFPI2 P48357 Leptin receptor LEPRP48594 Serpin B4 SERPINB4 P48645 Neuromedin-U-25 NMU P48740Mannan-binding lectin serine protease 1 MASP1 P48745 Protein NOV homologNOV P48960 CD97 antigen subunit beta CD97 P49223 Kunitz-type proteaseinhibitor 3 SPINT3 P49747 Cartilage oligomeric matrix protein COMPP49763 Placenta growth factor PGF P49765 Vascular endothelial growthfactor B VEGFB P49767 Vascular endothelial growth factor C VEGFC P49771Fms-related tyrosine kinase 3 ligand FLT3LG P49862 Kallikrein-7 KLK7P49863 Granzyme K GZMK P49908 Selenoprotein P SEPP1 P49913 Antibacterialprotein FALL-39 CAMP P50607 Tubby protein homolog TUB P51124 Granzyme MGZMM P51512 Matrix metalloproteinase-16 MMP16 P51654 Glypican-3 GPC3P51671 Eotaxin CCL11 P51884 Lumican LUM P51888 Prolargin PRELP P52798Ephrin-A4 EFNA4 P52823 Stanniocalcin-1 STC1 P53420 Collagen alpha-4(IV)chain COL4A4 P53621 Coatomer subunit alpha COPA P54108 Cysteine-richsecretory protein 3 CRISP3 P54315 Pancreatic lipase-related protein 1PNLIPRP1 P54317 Pancreatic lipase-related protein 2 PNLIPRP2 P54793Arylsulfatase F ARSF P55000 Secreted Ly-6/uPAR-related protein 1 SLURP1P55001 Microfibrillar-associated protein 2 MFAP2 P55056 ApolipoproteinC-IV APOC4 P55058 Phospholipid transfer protein PLTP P55075 Fibroblastgrowth factor 8 FGF8 P55081 Microfibrillar-associated protein 1 MFAP1P55083 Microfibril-associated glycoprotein 4 MFAP4 P55107 Bonemorphogenetic protein 3B GDF10 P55145 Mesencephalic astrocyte-derivedneurotrophic MANF factor P55259 Pancreatic secretory granule membranemajor GP2 glycoprotein GP2 P55268 Laminin subunit beta-2 LAMB2 P55773CCL23(30-99) CCL23 P55774 C-C motif chemokine 18 CCL18 P55789 FAD-linkedsulfhydryl oxidase ALR GFER P56703 Proto-oncogene Wnt-3 WNT3 P56704Protein Wnt-3a WNT3A P56705 Protein Wnt-4 WNT4 P56706 Protein Wnt-7bWNT7B P56730 Neurotrypsin PRSS12 P56851 Epididymal secretory proteinE3-beta EDDM3B P56975 Neuregulin-3 NRG3 P58062 Serine protease inhibitorKazal-type 7 SPINK7 P58215 Lysyl oxidase homolog 3 LOXL3 P58294Prokineticin-1 PROK1 P58335 Anthrax toxin receptor 2 ANTXR2 P58397 Adisintegrin and metalloproteinase with ADAMTS12 thrombospondin motifs 12P58417 Neurexophilin-1 NXPH1 P58499 Protein FAM3B FAM3B P59510 Adisintegrin and metalloproteinase with ADAMTS20 thrombospondin motifs 20P59665 Neutrophil defensin 1 DEFA1B P59666 Neutrophil defensin 3 DEFA3P59796 Glutathione peroxidase 6 GPX6 P59826 BPI fold-containing family Bmember 3 BPIFB3 P59827 BPI fold-containing family B member 4 BPIFB4P59861 Beta-defensin 131 DEFB131 P60022 Beta-defensin 1 DEFB1 P60153Inactive ribonuclease-like protein 9 RNASE9 P60827 Complement C1q tumornecrosis factor-related C1QTNF8 protein 8 P60852 Zona pellucidasperm-binding protein 1 ZP1 P60985 Keratinocytedifferentiation-associated protein KRTDAP P61109 Kidneyandrogen-regulated protein KAP P61278 Somatostatin-14 SST P61366Osteocrin OSTN P61626 Lysozyme C LYZ P61769 Beta-2-microglobulin B2MP61812 Transforming growth factor beta-2 TGFB2 P61916 Epididymalsecretory protein E1 NPC2 P62502 Epididymal-specific lipocalin-6 LCN6P62937 Peptidyl-prolyl cis-trans isomerase A PPIA P67809Nuclease-sensitive element-binding protein 1 YBX1 P67812 Signalpeptidase complex catalytic subunit SEC11A SEC11A P78310 Coxsackievirusand adenovirus receptor CXADR P78333 Secreted glypican-5 GPC5 P78380Oxidized low-density lipoprotein receptor 1 OLR1 P78423 Processedfractalkine CX3CL1 P78509 Reelin RELN P78556 CCL20(2-70) CCL20 P80075MCP-2(6-76) CCL8 P80098 C-C motif chemokine 7 CCL7 P80108Phosphatidylinositol-glycan-specific GPLD1 phospholipase D P80162 C—X—Cmotif chemokine 6 CXCL6 P80188 Neutrophil gelatinase-associatedlipocalin LCN2 P80303 Nucleobindin-2 NUCB2 P80511 Calcitermin S100A12P81172 Hepcidin-25 HAMP P81277 Prolactin-releasing peptide PRLH P81534Beta-defensin 103 DEFB103A P81605 Dermcidin DCD P82279 Protein crumbshomolog 1 CRB1 P82987 ADAMTS-like protein 3 ADAMTSL3 P83105 Serineprotease HTRA4 HTRA4 P83110 Serine protease HTRA3 HTRA3 P83859Orexigenic neuropeptide QRFP QRFP P98088 Mucin-5AC MUC5AC P98095Fibulin-2 FBLN2 P98160 Basement membrane-specific heparan sulfate HSPG2proteoglycan core protein P98173 Protein FAM3A FAM3A Q00604 Norrin NDPQ00796 Sorbitol dehydrogenase SORD Q00887 Pregnancy-specificbeta-1-glycoprotein 9 PSG9 Q00888 Pregnancy-specific beta-1-glycoprotein4 PSG4 Q00889 Pregnancy-specific beta-1-glycoprotein 6 PSG6 Q01523HD5(56-94) DEFA5 Q01524 Defensin-6 DEFA6 Q01955 Collagen alpha-3(IV)chain COL4A3 Q02297 Pro-neuregulin-1, membrane-bound isoform NRG1 Q02325Plasminogen-like protein B PLGLB1 Q02383 Semenogelin-2 SEMG2 Q02388Collagen alpha-1(VII) chain COL7A1 Q02505 Mucin-3A MUC3A Q02509Otoconin-90 OC90 Q02747 Guanylin GUCA2A Q02763 Angiopoietin-1 receptorTEK Q02817 Mucin-2 MUC2 Q02985 Complement factor H-related protein 3CFHR3 Q03167 Transforming growth factor beta receptor type 3 TGFBR3Q03403 Trefoil factor 2 TFF2 Q03405 Urokinase plasminogen activatorsurface receptor PLAUR Q03591 Complement factor H-related protein 1CFHR1 Q03692 Collagen alpha-1(X) chain COL10A1 Q04118 Basic salivaryproline-rich protein 3 PRB3 Q04756 Hepatocyte growth factor activatorshort chain HGFAC Q04900 Sialomucin core protein 24 CD164 Q05315Eosinophil lysophospholipase CLC Q05707 Collagen alpha-1(XIV) chainCOL14A1 Q05996 Processed zona pellucida sperm-binding protein 2 ZP2Q06033 Inter-alpha-trypsin inhibitor heavy chain H3 ITIH3 Q06141Regenerating islet-derived protein 3-alpha REG3A Q06828 FibromodulinFMOD Q07092 Collagen alpha-1(XVI) chain COL16A1 Q07325 C—X—C motifchemokine 9 CXCL9 Q07507 Dermatopontin DPT Q075Z2 Binder of spermprotein homolog 1 BSPH1 Q07654 Trefoil factor 3 TFF3 Q07699 Sodiumchannel subunit beta-1 SCN1B Q08345 Epithelial discoidindomain-containing receptor 1 DDR1 Q08380 Galectin-3-binding proteinLGALS3BP Q08397 Lysyl oxidase homolog 1 LOXL1 Q08431 Lactadherin MFGE8Q08629 Testican-1 SPOCK1 Q08648 Sperm-associated antigen 11B SPAG11BQ08830 Fibrinogen-like protein 1 FGL1 Q10471 PolypeptideN-acetylgalactosaminyltransferase 2 GALNT2 Q10472 PolypeptideN-acetylgalactosaminyltransferase 1 GALNT1 Q11201CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL1alpha-2,3-sialyltransferase 1 Q11203CMP-N-acetylneuraminate-beta-1,4-galactoside ST3GAL3alpha-2,3-sialyltransferase Q11206CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL4alpha-2,3-sialyltransferase 4 Q12794 Hyaluronidase-1 HYAL1 Q12805EGF-containing fibulin-like extracellular matrix EFEMP1 protein 1 Q12836Zona pellucida sperm-binding protein 4 ZP4 Q12841 Follistatin-relatedprotein 1 FSTL1 Q12904 Aminoacyl tRNA synthase complex-interacting AIMP1multifunctional protein 1 Q13018 Soluble secretory phospholipase A2receptor PLA2R1 Q13072 B melanoma antigen 1 BAGE Q13093Platelet-activating factor acetylhydrolase PLA2G7 Q13103 Secretedphosphoprotein 24 SPP2 Q13162 Peroxiredoxin-4 PRDX4 Q13201 Plateletglycoprotein Ia* MMRN1 Q13214 Semaphorin-3B SEMA3B Q13219 Pappalysin-1PAPPA Q13231 Chitotriosidase-1 CHIT1 Q13253 Noggin NOG Q13261Interleukin-15 receptor subunit alpha IL15RA Q13275 Semaphorin-3F SEMA3FQ13291 Signaling lymphocytic activation molecule SLAMF1 Q13316 Dentinmatrix acidic phosphoprotein 1 DMP1 Q13361 Microfibrillar-associatedprotein 5 MFAP5 Q13410 Butyrophilin subfamily 1 member A1 BTN1A1 Q13421Mesothelin, cleaved form MSLN Q13429 Insulin-like growth factor I IGF-IQ13443 Disintegrin and metalloproteinase domain- ADAM9 containingprotein 9 Q13519 Neuropeptide 1 PNOC Q13751 Laminin subunit beta-3 LAMB3Q13753 Laminin subunit gamma-2 LAMC2 Q13790 Apolipoprotein F APOF Q13822Ectonucleotide ENPP2 pyrophosphatase/phosphodiesterase family member 2Q14031 Collagen alpha-6(IV) chain COL4A6 Q14050 Collagen alpha-3(IX)chain COL9A3 Q14055 Collagen alpha-2(IX) chain COL9A2 Q14112 Nidogen-2NID2 Q14114 Low-density lipoprotein receptor-related protein 8 LRP8Q14118 Dystroglycan DAG1 Q14314 Fibroleukin FGL2 Q14393 Growtharrest-specific protein 6 GAS6 Q14406 Chorionic somatomammotropinhormone-like 1 CSHL1 Q14507 Epididymal secretory protein E3-alpha EDDM3AQ14508 WAP four-disulfide core domain protein 2 WFDC2 Q14512 Fibroblastgrowth factor-binding protein 1 FGFBP1 Q14515 SPARC-like protein 1SPARCL1 Q14520 Hyaluronan-binding protein 2 27 kDa light chain HABP2Q14563 Semaphorin-3A SEMA3A Q14623 Indian hedgehog protein IHH Q14624Inter-alpha-trypsin inhibitor heavy chain H4 ITIH4 Q14667 UPF0378protein KIAA0100 KIAA0100 Q14703 Membrane-bound transcription factorsite-1 MBTPS1 protease Q14766 Latent-transforming growth factorbeta-binding LTBP1 protein 1 Q14767 Latent-transforming growth factorbeta-binding LTBP2 protein 2 Q14773 Intercellular adhesion molecule 4ICAM4 Q14993 Collagen alpha-1(XIX) chain COL19A1 Q14CN2Calcium-activated chloride channel regulator 4, CLCA4 110 kDa formQ15046 Lysine--tRNA ligase KARS Q15063 Periostin POSTN Q15109 Advancedglycosylation end product-specific AGER receptor Q15113 ProcollagenC-endopeptidase enhancer 1 PCOLCE Q15166 Serum paraoxonase/lactonase 3PON3 Q15195 Plasminogen-like protein A PLGLA Q15198 Platelet-derivedgrowth factor receptor-like protein PDGFRL Q15223 Poliovirusreceptor-related protein 1 PVRL1 Q15238 Pregnancy-specificbeta-1-glycoprotein 5 PSG5 Q15363 Transmembrane emp24 domain-containingprotein 2 TMED2 Q15375 Ephrin type-A receptor 7 EPHA7 Q15389Angiopoietin-1 ANGPT1 Q15465 Sonic hedgehog protein SHH Q15485 Ficolin-2FCN2 Q15517 Corneodesmosin CDSN Q15582 Transforming growthfactor-beta-induced protein TGFBI ig-h3 Q15661 Tryptase alpha/beta-1TPSAB1 Q15726 Metastin KISS1 Q15782 Chitinase-3-like protein 2 CHI3L2Q15828 Cystatin-M CST6 Q15846 Clusterin-like protein 1 CLUL1 Q15848Adiponectin ADIPOQ Q16206 Protein disulfide-thiol oxidoreductase ENOX2Q16270 Insulin-like growth factor-binding protein 7 IGFBP7 Q16363Laminin subunit alpha-4 LAMA4 Q16378 Proline-rich protein 4 PRR4 Q16557Pregnancy-specific beta-1-glycoprotein 3 PSG3 Q16568 CART(42-89) CARTPTQ16610 Extracellular matrix protein 1 ECM1 Q16619 Cardiotrophin-1 CTF1Q16623 Syntaxin-1A STX1A Q16627 HCC-1(9-74) CCL14 Q16651 Prostasin lightchain PRSS8 Q16661 Guanylate cyclase C-activating peptide 2 GUCA2BQ16663 CCL15(29-92) CCL15 Q16674 Melanoma-derived growth regulatoryprotein MIA Q16769 Glutaminyl-peptide cyclotransferase QPCT Q16787Laminin subunit alpha-3 LAMA3 Q16842CMP-N-acetylneuraminate-beta-galactosamide- ST3GAL2alpha-2,3-sialyltransferase 2 Q17RR3 Pancreatic lipase-related protein 3PNLIPRP3 Q17RW2 Collagen alpha-1(XXIV) chain COL24A1 Q17RY6 Lymphocyteantigen 6K LY6K Q1L6U9 Prostate-associated microseminoprotein MSMPQ1W4C9 Serine protease inhibitor Kazal-type 13 SPINK13 Q1ZYL8 Izumosperm-egg fusion protein 4 IZUMO4 Q29960 HLA class I histocompatibilityantigen, Cw-16 HLA-C alpha chain Q2I0M5 R-spondin-4 RSPO4 Q2L4Q9 Serineprotease 53 PRSS53 Q2MKA7 R-spondin-1 RSPO1 Q2MV58 Tectonic-1 TCTN1Q2TAL6 Brorin VWC2 Q2UY09 Collagen alpha-1(XXVIII) chain COL28A1 Q2VPA4Complement component receptor 1-like protein CR1L Q2WEN9Carcinoembryonic antigen-related cell adhesion CEACAM16 molecule 16Q30KP8 Beta-defensin 136 DEFB136 Q30KP9 Beta-defensin 135 DEFB135 Q30KQ1Beta-defensin 133 DEFB133 Q30KQ2 Beta-defensin 130 DEFB130 Q30KQ4Beta-defensin 116 DEFB116 Q30KQ5 Beta-defensin 115 DEFB115 Q30KQ6Beta-defensin 114 DEFB114 Q30KQ7 Beta-defensin 113 DEFB113 Q30KQ8Beta-defensin 112 DEFB112 Q30KQ9 Beta-defensin 110 DEFB110 Q30KR1Beta-defensin 109 DEFB109P1 Q32P28 Prolyl 3-hydroxylase 1 LEPRE1 Q3B7J2Glucose-fructose oxidoreductase domain- GFOD2 containing protein 2Q3SY79 Protein Wnt WNT3A Q3T906 N-acetylglucosamine-1-phosphotransferaseGNPTAB subunits alpha/beta Q495T6 Membrane metallo-endopeptidase-like 1MMEL1 Q49AH0 Cerebral dopamine neurotrophic factor CDNF Q4G0G5Secretoglobin family 2B member 2 SCGB2B2 Q4G0M1 Protein FAM132B FAM132BQ4LDE5 Sushi, von Willebrand factor type A, EGF and SVEP1 pentraxindomain-containing protein 1 Q4QY38 Beta-defensin 134 DEFB134 Q4VAJ4Protein Wnt WNT10B Q4W5P6 Protein TMEM155 TMEM155 Q4ZHG4 Fibronectintype III domain-containing protein 1 FNDC1 Q53H76 Phospholipase A1member A PLA1A Q53RD9 Fibulin-7 FBLN7 Q53S33 BolA-like protein 3 BOLA3Q5BLP8 Neuropeptide-like protein C4orf48 C4orf48 Q5DT21 Serine proteaseinhibitor Kazal-type 9 SPINK9 Q5EBL8 PDZ domain-containing protein 11PDZD11 Q5FYB0 Arylsulfatase J ARSJ Q5FYB1 Arylsulfatase I ARSI Q5GAN3Ribonuclease-like protein 13 RNASE13 Q5GAN4 Ribonuclease-like protein 12RNASE12 Q5GAN6 Ribonuclease-like protein 10 RNASE10 Q5GFL6 vonWillebrand factor A domain-containing VWA2 protein 2 Q5H8A3 Neuromedin-SNMS Q5H8C1 FRAS1-related extracellular matrix protein 1 FREM1 Q5IJ48Protein crumbs homolog 2 CRB2 Q5J5C9 Beta-defensin 121 DEFB121 Q5JS37NHL repeat-containing protein 3 NHLRC3 Q5JTB6 Placenta-specific protein9 PLAC9 Q5JU69 Torsin-2A TOR2A Q5JXM2 Methyltransferase-like protein 24METTL24 Q5JZY3 Ephrin type-A receptor 10 EPHA10 Q5K4E3 Polyserase-2PRSS36 Q5SRR4 Lymphocyte antigen 6 complex locus protein G5c LY6G5CQ5T1H1 Protein eyes shut homolog EYS Q5T4F7 Secreted frizzled-relatedprotein 5 SFRP5 Q5T4W7 Artemin ARTN Q5T7M4 Protein FAM132A FAM132AQ5TEH8 Protein Wnt WNT2B Q5TIE3 von Willebrand factor Adomain-containing VWA5B1 protein 5B1 Q5UCC4 ER membrane protein complexsubunit 10 EMC10 Q5VST6 Abhydrolase domain-containing protein FAM108B1FAM108B1 Q5VTL7 Fibronectin type III domain-containing protein 7 FNDC7Q5VUM1 UPF0369 protein C6orf57 C6orf57 Q5VV43 Dyslexia-associatedprotein KIAA0319 KIAA0319 Q5VWW1 Complement C1q-like protein 3 C1QL3Q5VXI9 Lipase member N LIPN Q5VXJ0 Lipase member K LIPK Q5VXM1 CUBdomain-containing protein 2 CDCP2 Q5VYX0 Renalase RNLS Q5VYY2 Lipasemember M LIPM Q5W186 Cystatin-9 CST9 Q5W5W9 Regulated endocrine-specificprotein 18 RESP18 Q5XG92 Carboxylesterase 4A CES4A Q63HQ2 PikachurinEGFLAM Q641Q3 Meteorin-like protein METRNL Q66K79 Carboxypeptidase Z CPZQ685J3 Mucin-17 MUC17 Q68BL7 Olfactomedin-like protein 2A OLFML2A Q68BL8Olfactomedin-like protein 2B OLFML2B Q68DV7 E3 ubiquitin-protein ligaseRNF43 RNF43 Q6B9Z1 Insulin growth factor-like family member 4 IGFL4Q6BAA4 Fc receptor-like B FCRLB Q6E0U4 Dermokine DMKN Q6EMK4 VasorinVASN Q6FHJ7 Secreted frizzled-related protein 4 SFRP4 Q6GPI1Chymotrypsin B2 chain B CTRB2 Q6GTS8 Probable carboxypeptidase PM20D1PM20D1 Q6H9L7 Isthmin-2 ISM2 Q6IE36 Ovostatin homolog 2 OVOS2 Q6IE37Ovostatin homolog 1 OVOS1 Q6IE38 Serine protease inhibitor Kazal-type 14SPINK14 Q6ISS4 Leukocyte-associated immunoglobulin-like LAIR2 receptor 2Q6JVE5 Epididymal-specific lipocalin-12 LCN12 Q6JVE6 Epididymal-specificlipocalin-10 LCN10 Q6JVE9 Epididymal-specific lipocalin-8 LCN8 Q6KF10Growth/differentiation factor 6 GDF6 Q6MZW2 Follistatin-related protein4 FSTL4 Q6NSX1 Coiled-coil domain-containing protein 70 CCDC70 Q6NT32Carboxylesterase 5A CES5A Q6NT52 Choriogonadotropin subunit beta variant2 CGB2 Q6NUI6 Chondroadherin-like protein CHADL Q6NUJ1 Saposin A-likePSAPL1 Q6P093 Arylacetamide deacetylase-like 2 AADACL2 Q6P4A8Phospholipase B-like 1 PLBD1 Q6P5S2 UPF0762 protein C6orf58 C6orf58Q6P988 Protein notum homolog NOTUM Q6PCB0 von Willebrand factor Adomain-containing VWA1 protein 1 Q6PDA7 Sperm-associated antigen 11ASPAG11A Q6PEW0 Inactive serine protease 54 PRSS54 Q6PEZ8 Podocan-likeprotein 1 PODNL1 Q6PKH6 Dehydrogenase/reductase SDR family member 4-DHRS4L2 like 2 Q6Q788 Apolipoprotein A-V APOA5 Q6SPF0 Atherin SAMD1Q6UDR6 Kunitz-type protease inhibitor 4 SPINT4 Q6URK8 Testis, prostateand placenta-expressed protein TEPP Q6UW01 Cerebellin-3 CBLN3 Q6UW10Surfactant-associated protein 2 SFTA2 Q6UW15 Regenerating islet-derivedprotein 3-gamma REG3G Q6UW32 Insulin growth factor-like family member 1IGFL1 Q6UW78 UPF0723 protein C11orf83 C11orf83 Q6UW88 Epigen EPGN Q6UWE3Colipase-like protein 2 CLPSL2 Q6UWF7 NXPE family member 4 NXPE4 Q6UWF9Protein FAM180A FAM180A Q6UWM5 GLIPR1-like protein 1 GLIPR1L1 Q6UWN8Serine protease inhibitor Kazal-type 6 SPINK6 Q6UWP2Dehydrogenase/reductase SDR family member 11 DHRS11 Q6UWP8 SuprabasinSBSN Q6UWQ5 Lysozyme-like protein 1 LYZL1 Q6UWQ7 Insulin growthfactor-like family member 2 IGFL2 Q6UWR7 Ectonucleotide ENPP6pyrophosphatase/phosphodiesterase family member 6 soluble form Q6UWT2Adropin ENHO Q6UWU2 Beta-galactosidase-1-like protein GLB1L Q6UWW0Lipocalin-15 LCN15 Q6UWX4 HHIP-like protein 2 HHIPL2 Q6UWY0Arylsulfatase K ARSK Q6UWY2 Serine protease 57 PRSS57 Q6UWY5Olfactomedin-like protein 1 OLFML1 Q6UX06 Olfactomedin-4 OLFM4 Q6UX07Dehydrogenase/reductase SDR family member 13 DHRS13 Q6UX39 Amelotin AMTNQ6UX46 Protein FAM150B FAM150B Q6UX73 UPF0764 protein C16orf89 C16orf89Q6UXB0 Protein FAM131A FAM131A Q6UXB1 Insulin growth factor-like familymember 3 IGFL3 Q6UXB2 VEGF co-regulated chemokine 1 CXCL17 Q6UXF7 C-typelectin domain family 18 member B CLEC18B Q6UXH0 Hepatocellularcarcinoma-associated protein TD26 C19orf80 Q6UXH1 Cysteine-rich withEGF-like domain protein 2 CRELD2 Q6UXH8 Collagen and calcium-binding EGFdomain- CCBE1 containing protein 1 Q6UXH9 Inactive serine protease PAMR1PAMR1 Q6UXI7 Vitrin VIT Q6UXI9 Nephronectin NPNT Q6UXN2 Trem-liketranscript 4 protein TREML4 Q6UXS0 C-type lectin domain family 19 memberA CLEC19A Q6UXT8 Protein FAM150A FAM150A Q6UXT9 Abhydrolasedomain-containing protein 15 ABHD15 Q6UXV4 Apolipoprotein O-like APOOLQ6UXX5 Inter-alpha-trypsin inhibitor heavy chain H6 ITIH6 Q6UXX9R-spondin-2 RSPO2 Q6UY14 ADAMTS-like protein 4 ADAMTSL4 Q6UY27 Prostateand testis expressed protein 2 PATE2 Q6W4X9 Mucin-6 MUC6 Q6WN34Chordin-like protein 2 CHRDL2 Q6WRI0 Immunoglobulin superfamily member10 IGSF10 Q6X4U4 Sclerostin domain-containing protein 1 SOSTDC1 Q6X784Zona pellucida-binding protein 2 ZPBP2 Q6XE38 Secretoglobin family 1Dmember 4 SCGB1D4 Q6XPR3 Repetin RPTN Q6XZB0 Lipase member I LIPI Q6ZMM2ADAMTS-like protein 5 ADAMTSL5 Q6ZMP0 Thrombospondin type-1domain-containing THSD4 protein 4 Q6ZNF0 Iron/zinc purple acidphosphatase-like protein PAPL Q6ZRI0 Otogelin OTOG Q6ZRP7 Sulfhydryloxidase 2 QSOX2 Q6ZWJ8 Kielin/chordin-like protein KCP Q75N90Fibrillin-3 FBN3 Q765I0 Urotensin-2B UTS2D Q76B58 Protein FAM5C FAM5CQ76LX8 A disintegrin and metalloproteinase with ADAMTS13 thrombospondinmotifs 13 Q76M96 Coiled-coil domain-containing protein 80 CCDC80 Q7L1S5Carbohydrate sulfotransferase 9 CHST9 Q7L513 Fc receptor-like A FCRLAQ7L8A9 Vasohibin-1 VASH1 Q7RTM1 Otopetrin-1 OTOP1 Q7RTW8 Otoancorin OTOAQ7RTY5 Serine protease 48 PRSS48 Q7RTY7 Ovochymase-1 OVCH1 Q7RTZ1Ovochymase-2 OVCH2 Q7Z304 MAM domain-containing protein 2 MAMDC2 Q7Z3S9Notch homolog 2 N-terminal-like protein NOTCH2NL Q7Z4H4 Intermedin-shortADM2 Q7Z4P5 Growth/differentiation factor 7 GDF7 Q7Z4R8 UPF0669 proteinC6orf120 C6orf120 Q7Z4W2 Lysozyme-like protein 2 LYZL2 Q7Z5A4 Serineprotease 42 PRSS42 Q7Z5A7 Protein FAM19A5 FAM19A5 Q7Z5A8 Protein FAM19A3FAM19A3 Q7Z5A9 Protein FAM19A1 FAM19A1 Q7Z5J1 Hydroxysteroid11-beta-dehydrogenase 1-like HSD11B1L protein Q7Z5L0 Vitelline membraneouter layer protein 1 homolog VMO1 Q7Z5L3 Complement C1q-like protein 2C1QL2 Q7Z5L7 Podocan PODN Q7Z5P4 17-beta-hydroxysteroid dehydrogenase 13HSD17B13 Q7Z5P9 Mucin-19 MUC19 Q7Z5Y6 Bone morphogenetic protein 8ABMP8A Q7Z7B7 Beta-defensin 132 DEFB132 Q7Z7B8 Beta-defensin 128 DEFB128Q7Z7C8 Transcription initiation factor TFIID subunit 8 TAF8 Q7Z7H5Transmembrane emp24 domain-containing protein 4 TMED4 Q86SG7 Lysozymeg-like protein 2 LYG2 Q86SI9 Protein CEI C5orf38 Q86TE4 Leucine zipperprotein 2 LUZP2 Q86TH1 ADAMTS-like protein 2 ADAMTSL2 Q86U17 Serpin A11SERPINA11 Q86UU9 Endokinin-A TAC4 Q86UW8 Hyaluronan and proteoglycanlink protein 4 HAPLN4 Q86UX2 Inter-alpha-trypsin inhibitor heavy chainH5 ITIH5 Q86V24 Adiponectin receptor protein 2 ADIPOR2 Q86VB7 SolubleCD163 CD163 Q86VR8 Four-jointed box protein 1 FJX1 Q86WD7 Serpin A9SERPINA9 Q86WN2 Interferon epsilon IFNE Q86WS3 Placenta-specific 1-likeprotein PLAC1L Q86X52 Chondroitin sulfate synthase 1 CHSY1 Q86XP6Gastrokine-2 GKN2 Q86XS5 Angiopoietin-related protein 5 ANGPTL5 Q86Y27 Bmelanoma antigen 5 BAGE5 Q86Y28 B melanoma antigen 4 BAGE4 Q86Y29 Bmelanoma antigen 3 BAGE3 Q86Y30 B melanoma antigen 2 BAGE2 Q86Y38Xylosyltransferase 1 XYLT1 Q86Y78 Ly6/PLAUR domain-containing protein 6LYPD6 Q86YD3 Transmembrane protein 25 TMEM25 Q86YJ6 Threoninesynthase-like 2 THNSL2 Q86YW7 Glycoprotein hormone beta-5 GPHB5 Q86Z23Complement C1q-like protein 4 C1QL4 Q8IU57 Interleukin-28 receptorsubunit alpha IL28RA Q8IUA0 WAP four-disulfide core domain protein 8WFDC8 Q8IUB2 WAP four-disulfide core domain protein 3 WFDC3 Q8IUB3Protein WFDC10B WFDC10B Q8IUB5 WAP four-disulfide core domain protein 13WFDC13 Q8IUH2 Protein CREG2 CREG2 Q8IUK5 Plexin domain-containingprotein 1 PLXDC1 Q8IUL8 Cartilage intermediate layer protein 2 C2 CILP2Q8IUX7 Adipocyte enhancer-binding protein 1 AEBP1 Q8IUX8 Epidermalgrowth factor-like protein 6 EGFL6 Q8IVL8 Carboxypeptidase O CPO Q8IVN8Somatomedin-B and thrombospondin type-1 SBSPON domain-containing proteinQ8IVW8 Protein spinster homolog 2 SPNS2 Q8IW75 Serpin A12 SERPINA12Q8IW92 Beta-galactosidase-1-like protein 2 GLB1L2 Q8IWL1 Pulmonarysurfactant-associated protein A2 SFTPA2 Q8IWL2 Pulmonarysurfactant-associated protein A1 SFTPA1 Q8IWV2 Contactin-4 CNTN4 Q8IWY4Signal peptide, CUB and EGF-like domain- SCUBE1 containing protein 1Q8IX30 Signal peptide, CUB and EGF-like domain- SCUBE3 containingprotein 3 Q8IXA5 Sperm acrosome membrane-associated protein 3, SPACA3membrane form Q8IXB1 DnaJ homolog subfamily C member 10 DNAJC10 Q8IXL6Extracellular serine/threonine protein kinase FAM20C Fam20C Q8IYD9 Lungadenoma susceptibility protein 2 LAS2 Q8IYP2 Serine protease 58 PRSS58Q8IYS5 Osteoclast-associated immunoglobulin-like OSCAR receptor Q8IZC6Collagen alpha-1(XXVII) chain COL27A1 Q8IZJ3 C3 and PZP-likealpha-2-macroglobulin domain- CPAMD8 containing protein 8 Q8IZN7Beta-defensin 107 DEFB107B Q8N0V4 Leucine-rich repeat LGI family member2 LGI2 Q8N104 Beta-defensin 106 DEFB106B Q8N119 Matrixmetalloproteinase-21 MMP21 Q8N129 Protein canopy homolog 4 CNPY4 Q8N135Leucine-rich repeat LGI family member 4 LGI4 Q8N145 Leucine-rich repeatLGI family member 3 LGI3 Q8N158 Glypican-2 GPC2 Q8N1E2 Lysozyme g-likeprotein 1 LYG1 Q8N2E2 von Willebrand factor D and EGF domain- VWDEcontaining protein Q8N2E6 Prosalusin TOR2A Q8N2S1 Latent-transforminggrowth factor beta-binding LTBP4 protein 4 Q8N302 Angiogenic factor withG patch and FHA domains 1 AGGF1 Q8N307 Mucin-20 MUC20 Q8N323 NXPE familymember 1 NXPE1 Q8N387 Mucin-15 MUC15 Q8N3Z0 Inactive serine protease 35PRSS35 Q8N436 Inactive carboxypeptidase-like protein X2 CPXM2 Q8N474Secreted frizzled-related protein 1 SFRP1 Q8N475 Follistatin-relatedprotein 5 FSTL5 Q8N4F0 BPI fold-containing family B member 2 BPIFB2Q8N4T0 Carboxypeptidase A6 CPA6 Q8N5W8 Protein FAM24B FAM24B Q8N687Beta-defensin 125 DEFB125 Q8N688 Beta-defensin 123 DEFB123 Q8N690Beta-defensin 119 DEFB119 Q8N6C5 Immunoglobulin superfamily member 1IGSF1 Q8N6C8 Leukocyte immunoglobulin-like receptor LILRA3 subfamily Amember 3 Q8N6G6 ADAMTS-like protein 1 ADAMTSL1 Q8N6Y2 Leucine-richrepeat-containing protein 17 LRRC17 Q8N729 Neuropeptide W-23 NPW Q8N8U9BMP-binding endothelial regulator protein BMPER Q8N907 DAN domain familymember 5 DAND5 Q8NAT1 Glycosyltransferase-like domain-containing GTDC2protein 2 Q8NAU1 Fibronectin type III domain-containing protein 5 FNDC5Q8NB37 Parkinson disease 7 domain-containing protein 1 PDDC1 Q8NBI3Draxin DRAXIN Q8NBM8 Prenylcysteine oxidase-like PCYOX1L Q8NBP7Proprotein convertase subtilisin/kexin type 9 PCSK9 Q8NBQ5 Estradiol17-beta-dehydrogenase 11 HSD17B11 Q8NBV8 Synaptotagmin-8 SYT8 Q8NCC3Group XV phospholipase A2 PLA2G15 Q8NCF0 C-type lectin domain family 18member C CLEC18C Q8NCW5 NAD(P)H-hydrate epimerase APOA1BP Q8NDA2Hemicentin-2 HMCN2 Q8NDX9 Lymphocyte antigen 6 complex locus protein G5bLY6G5B Q8NDZ4 Deleted in autism protein 1 C3orf58 Q8NEB7 Acrosin-bindingprotein ACRBP Q8NES8 Beta-defensin 124 DEFB124 Q8NET1 Beta-defensin 108BDEFB108B Q8NEX5 Protein WFDC9 WFDC9 Q8NEX6 Protein WFDC11 WFDC11 Q8NF86Serine protease 33 PRSS33 Q8NFM7 Interleukin-17 receptor D IL17RD Q8NFQ5BPI fold-containing family B member 6 BPIFB6 Q8NFQ6 BPI fold-containingfamily C protein BPIFC Q8NFU4 Follicular dendritic cell secreted peptideFDCSP Q8NFW1 Collagen alpha-1(XXII) chain COL22A1 Q8NG35 Beta-defensin105 DEFB105B Q8NG41 Neuropeptide B-23 NPB Q8NHW6 Otospiralin OTOS Q8NI99Angiopoietin-related protein 6 ANGPTL6 Q8TAA1 Probable ribonuclease 11RNASE11 Q8TAG5 V-set and transmembrane domain-containing VSTM2A protein2A Q8TAL6 Fin bud initiation factor homolog FIBIN Q8TAT2 Fibroblastgrowth factor-binding protein 3 FGFBP3 Q8TAX7 Mucin-7 MUC7 Q8TB22Spermatogenesis-associated protein 20 SPATA20 Q8TB73 Protein NDNF NDNFQ8TB96 T-cell immunomodulatory protein ITFG1 Q8TC92 Proteindisulfide-thiol oxidoreductase ENOX1 Q8TCV5 WAP four-disulfide coredomain protein 5 WFDC5 Q8TD06 Anterior gradient protein 3 homolog AGR3Q8TD33 Secretoglobin family 1C member 1 SCGB1C1 Q8TD46 Cell surfaceglycoprotein CD200 receptor 1 CD200R1 Q8TDE3 Ribonuclease 8 RNASE8Q8TDF5 Neuropilin and tolloid-like protein 1 NETO1 Q8TDL5 BPIfold-containing family B member 1 BPIFB1 Q8TE56 A disintegrin andmetalloproteinase with ADAMTS17 thrombospondin motifs 17 Q8TE57 Adisintegrin and metalloproteinase with ADAMTS16 thrombospondin motifs 16Q8TE58 A disintegrin and metalloproteinase with ADAMTS15 thrombospondinmotifs 15 Q8TE59 A disintegrin and metalloproteinase with ADAMTS19thrombospondin motifs 19 Q8TE60 A disintegrin and metalloproteinase withADAMTS18 thrombospondin motifs 18 Q8TE99 Acid phosphatase-like protein 2ACPL2 Q8TER0 Sushi, nidogen and EGF-like domain-containing SNED1 protein1 Q8TEU8 WAP, kazal, immunoglobulin, kunitz and NTR WFIKKN2domain-containing protein 2 Q8WTQ1 Beta-defensin 104 DEFB104B Q8WTR8Netrin-5 NTN5 Q8WTU2 Scavenger receptor cysteine-rich domain- SRCRB4Dcontaining group B protein Q8WU66 Protein TSPEAR TSPEAR Q8WUA8 TsukushinTSKU Q8WUF8 Protein FAM172A FAM172A Q8WUJ1 Neuferricin CYB5D2 Q8WUY1UPF0670 protein THEM6 THEM6 Q8WVN6 Secreted and transmembrane protein 1SECTM1 Q8WVQ1 Soluble calcium-activated nucleotidase 1 CANT1 Q8WWA0Intelectin-1 ITLN1 Q8WWG1 Neuregulin-4 NRG4 Q8WWQ2 Inactive heparanase-2HPSE2 Q8WWU7 Intelectin-2 ITLN2 Q8WWY7 WAP four-disulfide core domainprotein 12 WFDC12 Q8WWY8 Lipase member H LIPH Q8WWZ8 Oncoprotein-inducedtranscript 3 protein OIT3 Q8WX39 Epididymal-specific lipocalin-9 LCN9Q8WXA2 Prostate and testis expressed protein 1 PATE1 Q8WXD2Secretogranin-3 SCG3 Q8WXF3 Relaxin-3 A chain RLN3 Q8WXI7 Mucin-16 MUC16Q8WXQ8 Carboxypeptidase A5 CPA5 Q8WXS8 A disintegrin andmetalloproteinase with ADAMTS14 thrombospondin motifs 14 Q92484 Acidsphingomyelinase-like phosphodiesterase 3a SMPDL3A Q92485 Acidsphingomyelinase-like phosphodiesterase 3b SMPDL3B Q92496 Complementfactor H-related protein 4 CFHR4 Q92520 Protein FAM3C FAM3C Q92563Testican-2 SPOCK2 Q92583 C-C motif chemokine 17 CCL17 Q92626 Peroxidasinhomolog PXDN Q92743 Serine protease HTRA1 HTRA1 Q92752 Tenascin-R TNRQ92765 Secreted frizzled-related protein 3 FRZB Q92819 Hyaluronansynthase 2 HAS2 Q92820 Gamma-glutamyl hydrolase GGH Q92824 Proproteinconvertase subtilisin/kexin type 5 PCSK5 Q92832 Protein kinase C-bindingprotein NELL1 NELL1 Q92838 Ectodysplasin-A, membrane form EDA Q92874Deoxyribonuclease-1-like 2 DNASE1L2 Q92876 Kallikrein-6 KLK6 Q92913Fibroblast growth factor 13 FGF13 Q92954 Proteoglycan 4 C-terminal partPRG4 Q93038 Tumor necrosis factor receptor superfamily TNFRSF25 member25 Q93091 Ribonuclease K6 RNASE6 Q93097 Protein Wnt-2b WNT2B Q93098Protein Wnt-8b WNT8B Q95460 Major histocompatibility complex classI-related MR1 gene protein Q969D9 Thymic stromal lymphopoietin TSLPQ969E1 Liver-expressed antimicrobial peptide 2 LEAP2 Q969H8 UPF0556protein C19orf10 C19orf10 Q969Y0 NXPE family member 3 NXPE3 Q96A54Adiponectin receptor protein 1 ADIPOR1 Q96A83 Collagen alpha-1(XXVI)chain EMID2 Q96A84 EMI domain-containing protein 1 EMID1 Q96A98Tuberoinfundibular peptide of 39 residues PTH2 Q96A99 Pentraxin-4 PTX4Q96BH3 Epididymal sperm-binding protein 1 ELSPBP1 Q96BQ1 Protein FAM3DFAM3D Q96CG8 Collagen triple helix repeat-containing protein 1 CTHRC1Q96DA0 Zymogen granule protein 16 homolog B ZG16B Q96DN2 von Willebrandfactor C and EGF domain- VWCE containing protein Q96DR5 BPIfold-containing family A member 2 BPIFA2 Q96DR8 Mucin-like protein 1MUCL1 Q96DX4 RING finger and SPRY domain-containing protein 1 RSPRY1Q96EE4 Coiled-coil domain-containing protein 126 CCDC126 Q96GS6Abhydrolase domain-containing protein FAM108A1 FAM108A1 Q96GW7 Brevicancore protein BCAN Q96HF1 Secreted frizzled-related protein 2 SFRP2Q96I82 Kazal-type serine protease inhibitor domain- KAZALD1 containingprotein 1 Q96ID5 Immunoglobulin superfamily member 21 IGSF21 Q96II8Leucine-rich repeat and calponin homology LRCH3 domain-containingprotein 3 Q96IY4 Carboxypeptidase B2 CPB2 Q96JB6 Lysyl oxidase homolog 4LOXL4 Q96JK4 HHIP-like protein 1 HHIPL1 Q96KN2 Beta-Ala-His dipeptidaseCNDP1 Q96KW9 Protein SPACA7 SPACA7 Q96KX0 Lysozyme-like protein 4 LYZL4Q96L15 Ecto-ADP-ribosyltransferase 5 ART5 Q96LB8 Peptidoglycanrecognition protein 4 PGLYRP4 Q96LB9 Peptidoglycan recognition protein 3PGLYRP3 Q96LC7 Sialic acid-binding Ig-like lectin 10 SIGLEC10 Q96LR4Protein FAM19A4 FAM19A4 Q96MK3 Protein FAM20A FAM20A Q96MS3Glycosyltransferase 1 domain-containing protein 1 GLT1D1 Q96NY8Processed poliovirus receptor-related protein 4 PVRL4 Q96NZ8 WAP, kazal,immunoglobulin, kunitz and NTR WFIKKN1 domain-containing protein 1Q96NZ9 Proline-rich acidic protein 1 PRAP1 Q96P44 Collagen alpha-1(XXI)chain COL21A1 Q96PB7 Noelin-3 OLFM3 Q96PC5 Melanoma inhibitory activityprotein 2 MIA2 Q96PD5 N-acetylmuramoyl-L-alanine amidase PGLYRP2 Q96PH6Beta-defensin 118 DEFB118 Q96PL1 Secretoglobin family 3A member 2SCGB3A2 Q96PL2 Beta-tectorin TECTB Q96QH8 Sperm acrosome-associatedprotein 5 SPACA5 Q96QR1 Secretoglobin family 3A member 1 SCGB3A1 Q96QU1Protocadherin-15 PCDH15 Q96QV1 Hedgehog-interacting protein HHIP Q96RW7Hemicentin-1 HMCN1 Q96S42 Nodal homolog NODAL Q96S86 Hyaluronan andproteoglycan link protein 3 HAPLN3 Q96SL4 Glutathione peroxidase 7 GPX7Q96SM3 Probable carboxypeptidase X1 CPXM1 Q96T91 Glycoprotein hormonealpha-2 GPHA2 Q99062 Granulocyte colony-stimulating factor receptorCSF3R Q99102 Mucin-4 alpha chain MUC4 Q99217 Amelogenin, X isoform AMELXQ99218 Amelogenin, Y isoform AMELY Q99435 Protein kinase C-bindingprotein NELL2 NELL2 Q99470 Stromal cell-derived factor 2 SDF2 Q99542Matrix metalloproteinase-19 MMP19 Q99574 Neuroserpin SERPINI1 Q99584Protein S100-A13 S100A13 Q99616 C-C motif chemokine 13 CCL13 Q99645Epiphycan EPYC Q99674 Cell growth regulator with EF hand domain CGREF1protein 1 Q99715 Collagen alpha-1(XII) chain COL12A1 Q99727Metalloproteinase inhibitor 4 TIMP4 Q99731 C-C motif chemokine 19 CCL19Q99748 Neurturin NRTN Q99935 Proline-rich protein 1 PROL1 Q99942 E3ubiquitin-protein ligase RNF5 RNF5 Q99944 Epidermal growth factor-likeprotein 8 EGFL8 Q99954 Submaxillary gland androgen-regulated protein 3ASMR3A Q99969 Retinoic acid receptor responder protein 2 RARRES2 Q99972Myocilin MYOC Q99983 Osteomodulin OMD Q99985 Semaphorin-3C SEMA3C Q99988Growth/differentiation factor 15 GDF15 Q9BPW4 Apolipoprotein L4 APOL4Q9BQ08 Resistin-like beta RETNLB Q9BQ16 Testican-3 SPOCK3 Q9BQ51Programmed cell death 1 ligand 2 PDCD1LG2 Q9BQB4 Sclerostin SOST Q9BQI4Coiled-coil domain-containing protein 3 CCDC3 Q9BQP9 BPI fold-containingfamily A member 3 BPIFA3 Q9BQR3 Serine protease 27 PRSS27 Q9BQY6 WAPfour-disulfide core domain protein 6 WFDC6 Q9BRR6 ADP-dependentglucokinase ADPGK Q9BS86 Zona pellucida-binding protein 1 ZPBP Q9BSG0Protease-associated domain-containing protein 1 PRADC1 Q9BSG5 RetbindinRTBDN Q9BT30 Probable alpha-ketoglutarate-dependent ALKBH7 dioxygenaseABH7 Q9BT56 Spexin C12orf39 Q9BT67 NEDD4 family-interacting protein 1NDFIP1 Q9BTY2 Plasma alpha-L-fucosidase FUCA2 Q9BU40 Chordin-likeprotein 1 CHRDL1 Q9BUD6 Spondin-2 SPON2 Q9BUN1 Protein MENT MENT Q9BUR5Apolipoprotein O APOO Q9BV94 ER degradation-enhancingalpha-mannosidase-like 2 EDEM2 Q9BWP8 Collectin-11 COLEC11 Q9BWS9Chitinase domain-containing protein 1 CHID1 Q9BX67 Junctional adhesionmolecule C JAM3 Q9BX93 Group XIIB secretory phospholipase A2-likePLA2G12B protein Q9BXI9 Complement C1q tumor necrosis factor-relatedC1QTNF6 protein 6 Q9BXJ0 Complement C1q tumor necrosis factor-relatedC1QTNF5 protein 5 Q9BXJ1 Complement C1q tumor necrosis factor-relatedC1QTNF1 protein 1 Q9BXJ2 Complement C1q tumor necrosis factor-relatedC1QTNF7 protein 7 Q9BXJ3 Complement C1q tumor necrosis factor-relatedC1QTNF4 protein 4 Q9BXJ4 Complement C1q tumor necrosis factor-relatedC1QTNF3 protein 3 Q9BXJ5 Complement C1q tumor necrosis factor-relatedC1QTNF2 protein 2 Q9BXN1 Asporin ASPN Q9BXP8 Pappalysin-2 PAPPA2 Q9BXR6Complement factor H-related protein 5 CFHR5 Q9BXS0 Collagen alpha-1(XXV)chain COL25A1 Q9BXX0 EMILIN-2 EMILIN2 Q9BXY4 R-spondin-3 RSPO3 Q9BY15EGF-like module-containing mucin-like hormone EMR3 receptor-like 3subunit beta Q9BY50 Signal peptidase complex catalytic subunit SEC11CSEC11C Q9BY76 Angiopoietin-related protein 4 ANGPTL4 Q9BYF1 Processedangiotensin-converting enzyme 2 ACE2 Q9BYJ0 Fibroblast growthfactor-binding protein 2 FGFBP2 Q9BYW3 Beta-defensin 126 DEFB126 Q9BYX4Interferon-induced helicase C domain-containing IFIH1 protein 1 Q9BYZ8Regenerating islet-derived protein 4 REG4 Q9BZ76 Contactin-associatedprotein-like 3 CNTNAP3 Q9BZG9 Ly-6/neurotoxin-like protein 1 LYNX1Q9BZJ3 Tryptase delta TPSD1 Q9BZM1 Group XIIA secretory phospholipase A2PLA2G12A Q9BZM2 Group IIF secretory phospholipase A2 PLA2G2F Q9BZM5NKG2D ligand 2 ULBP2 Q9BZP6 Acidic mammalian chitinase CHIA Q9BZZ2Sialoadhesin SIGLEC1 Q9C0B6 Protein FAM5B FAM5B Q9GZM7Tubulointerstitial nephritis antigen-like TINAGL1 Q9GZN4 Brain-specificserine protease 4 PRSS22 Q9GZP0 Platelet-derived growth factor D,receptor-binding PDGFD form Q9GZT5 Protein Wnt-10a WNT10A Q9GZU5Nyctalopin NYX Q9GZV7 Hyaluronan and proteoglycan link protein 2 HAPLN2Q9GZV9 Fibroblast growth factor 23 FGF23 Q9GZX9 Twisted gastrulationprotein homolog 1 TWSG1 Q9GZZ7 GDNF family receptor alpha-4 GFRA4 Q9GZZ8Extracellular glycoprotein lacritin LACRT Q9H0B8 Cysteine-rich secretoryprotein LCCL domain- CRISPLD2 containing 2 Q9H106 Signal-regulatoryprotein delta SIRPD Q9H114 Cystatin-like 1 CSTL1 Q9H173 Nucleotideexchange factor SIL1 SIL1 Q9H1E1 Ribonuclease 7 RNASE7 Q9H1F0 WAPfour-disulfide core domain protein 10A WFDC10A Q9H1J5 Protein Wnt-8aWNT8A Q9H1J7 Protein Wnt-5b WNT5B Q9H1M3 Beta-defensin 129 DEFB129Q9H1M4 Beta-defensin 127 DEFB127 Q9H1Z8 Augurin C2orf40 Q9H239 Matrixmetalloproteinase-28 MMP28 Q9H2A7 C—X—C motif chemokine 16 CXCL16 Q9H2A9Carbohydrate sulfotransferase 8 CHST8 Q9H2R5 Kallikrein-15 KLK15 Q9H2X0Chordin CHRD Q9H2X3 C-type lectin domain family 4 member M CLEC4M Q9H306Matrix metalloproteinase-27 MMP27 Q9H324 A disintegrin andmetalloproteinase with ADAMTS10 thrombospondin motifs 10 Q9H336Cysteine-rich secretory protein LCCL domain- CRISPLD1 containing 1Q9H3E2 Sorting nexin-25 SNX25 Q9H3R2 Mucin-13 MUC13 Q9H3U7 SPARC-relatedmodular calcium-binding protein 2 SMOC2 Q9H3Y0 Peptidase inhibitorR3HDML R3HDML Q9H4A4 Aminopeptidase B RNPEP Q9H4F8 SPARC-related modularcalcium-binding protein 1 SMOC1 Q9H4G1 Cystatin-9-like CST9L Q9H5V8 CUBdomain-containing protein 1 CDCP1 Q9H6B9 Epoxide hydrolase 3 EPHX3Q9H6E4 Coiled-coil domain-containing protein 134 CCDC134 Q9H741 UPF0454protein C12orf49 C12orf49 Q9H772 Gremlin-2 GREM2 Q9H7Y0 Deleted inautism-related protein 1 CXorf36 Q9H8L6 Multimerin-2 MMRN2 Q9H9S5Fukutin-related protein FKRP Q9HAT2 Sialate O-acetylesterase SIAE Q9HB40Retinoid-inducible serine carboxypeptidase SCPEP1 Q9HB63 Netrin-4 NTN4Q9HBJ0 Placenta-specific protein 1 PLAC1 Q9HC23 Prokineticin-2 PROK2Q9HC57 WAP four-disulfide core domain protein 1 WFDC1 Q9HC73 Cytokinereceptor-like factor 2 CRLF2 Q9HC84 Mucin-5B MUC5B Q9HCB6 Spondin-1SPON1 Q9HCQ7 Neuropeptide NPSF NPVF Q9HCT0 Fibroblast growth factor 22FGF22 Q9HD89 Resistin RETN Q9NNX1 Tuftelin TUFT1 Q9NNX6 CD209 antigenCD209 Q9NP55 BPI fold-containing family A member 1 BPIFA1 Q9NP70Ameloblastin AMBN Q9NP95 Fibroblast growth factor 20 FGF20 Q9NP99Triggering receptor expressed on myeloid cells 1 TREM1 Q9NPA2 Matrixmetalloproteinase-25 MMP25 Q9NPE2 Neugrin NGRN Q9NPH0 Lysophosphatidicacid phosphatase type 6 ACP6 Q9NPH6 Odorant-binding protein 2b OBP2BQ9NQ30 Endothelial cell-specific molecule 1 ESM1 Q9NQ36 Signal peptide,CUB and EGF-like domain- SCUBE2 containing protein 2 Q9NQ38 Serineprotease inhibitor Kazal-type 5 SPINK5 Q9NQ76 Matrix extracellularphosphoglycoprotein MEPE Q9NQ79 Cartilage acidic protein 1 CRTAC1 Q9NR16Scavenger receptor cysteine-rich type 1 protein CD163L1 M160 Q9NR23Growth/differentiation factor 3 GDF3 Q9NR71 Neutral ceramidase ASAH2Q9NR99 Matrix-remodeling-associated protein 5 MXRA5 Q9NRA1Platelet-derived growth factor C PDGFC Q9NRC9 Otoraplin OTOR Q9NRE1Matrix metalloproteinase-26 MMP26 Q9NRJ3 C-C motif chemokine 28 CCL28Q9NRM1 Enamelin ENAM Q9NRN5 Olfactomedin-like protein 3 OLFML3 Q9NRR1Cytokine-like protein 1 CYTL1 Q9NS15 Latent-transforming growth factorbeta-binding LTBP3 protein 3 Q9NS62 Thrombospondin type-1domain-containing THSD1 protein 1 Q9NS71 Gastrokine-1 GKN1 Q9NS98Semaphorin-3G SEMA3G Q9NSA1 Fibroblast growth factor 21 FGF21 Q9NT22EMILIN-3 EMILIN3 Q9NTU7 Cerebellin-4 CBLN4 Q9NVR0 Kelch-like protein 11KLHL11 Q9NWH7 Spermatogenesis-associated protein 6 SPATA6 Q9NXC2Glucose-fructose oxidoreductase domain- GFOD1 containing protein 1Q9NY56 Odorant-binding protein 2a OBP2A Q9NY84 Vascular non-inflammatorymolecule 3 VNN3 Q9NZ20 Group 3 secretory phospholipase A2 PLA2G3 Q9NZC2Triggering receptor expressed on myeloid cells 2 TREM2 Q9NZK5 Adenosinedeaminase CECR1 CECR1 Q9NZK7 Group IIE secretory phospholipase A2PLA2G2E Q9NZP8 Complement C1r subcomponent-like protein C1RL Q9NZV1Cysteine-rich motor neuron 1 protein CRIM1 Q9NZW4 Dentin sialoproteinDSPP Q9P0G3 Kallikrein-14 KLK14 Q9P0W0 Interferon kappa IFNK Q9P218Collagen alpha-1(XX) chain COL20A1 Q9P2C4 Transmembrane protein 181TMEM181 Q9P2K2 Thioredoxin domain-containing protein 16 TXNDC16 Q9P2N4 Adisintegrin and metalloproteinase with ADAMTS9 thrombospondin motifs 9Q9UBC7 Galanin-like peptide GALP Q9UBD3 Cytokine SCM-1 beta XCL2 Q9UBD9Cardiotrophin-like cytokine factor 1 CLCF1 Q9UBM4 Opticin OPTC Q9UBP4Dickkopf-related protein 3 DKK3 Q9UBQ6 Exostosin-like 2 EXTL2 Q9UBR5Chemokine-like factor CKLF Q9UBS5 Gamma-aminobutyric acid type Breceptor subunit 1 GABBR1 Q9UBT3 Dickkopf-related protein 4 short formDKK4 Q9UBU2 Dickkopf-related protein 2 DKK2 Q9UBU3 Ghrelin-28 GHRLQ9UBV4 Protein Wnt-16 WNT16 Q9UBX5 Fibulin-5 FBLN5 Q9UBX7 Kallikrein-11KLK11 Q9UEF7 Klotho KL Q9UFP1 Protein FAM198A FAM198A Q9UGM3 Deleted inmalignant brain tumors 1 protein DMBT1 Q9UGM5 Fetuin-B FETUB Q9UGP8Translocation protein SEC63 homolog SEC63 Q9UHF0 Neurokinin-B TAC3Q9UHF1 Epidermal growth factor-like protein 7 EGFL7 Q9UHG2 ProSAASPCSK1N Q9UHI8 A disintegrin and metalloproteinase with ADAMTS1thrombospondin motifs 1 Q9UHL4 Dipeptidyl peptidase 2 DPP7 Q9UI42Carboxypeptidase A4 CPA4 Q9UIG4 Psoriasis susceptibility 1 candidategene 2 protein PSORS1C2 Q9UIK5 Tomoregulin-2 TMEFF2 Q9UIQ6Leucyl-cystinyl aminopeptidase, pregnancy serum LNPEP form Q9UJA9Ectonucleotide ENPP5 pyrophosphatase/phosphodiesterase family member 5Q9UJH8 Meteorin METRN Q9UJJ9 N-acetylglucosamine-1-phosphotransferaseGNPTG subunit gamma Q9UJW2 Tubulointerstitial nephritis antigen TINAGQ9UK05 Growth/differentiation factor 2 GDF2 Q9UK55 Protein Z-dependentprotease inhibitor SERPINA10 Q9UK85 Dickkopf-like protein 1 DKKL1 Q9UKJ1Paired immunoglobulin-like type 2 receptor alpha PILRA Q9UKP4 Adisintegrin and metalloproteinase with ADAMTS7 thrombospondin motifs 7Q9UKP5 A disintegrin and metalloproteinase with ADAMTS6 thrombospondinmotifs 6 Q9UKQ2 Disintegrin and metalloproteinase domain- ADAM28containing protein 28 Q9UKQ9 Kallikrein-9 KLK9 Q9UKR0 Kallikrein-12KLK12 Q9UKR3 Kallikrein-13 KLK13 Q9UKU9 Angiopoietin-related protein 2ANGPTL2 Q9UKZ9 Procollagen C-endopeptidase enhancer 2 PCOLCE2 Q9UL52Transmembrane protease serine 11E non-catalytic TMPRSS11E chain Q9ULC0Endomucin EMCN Q9ULI3 Protein HEG homolog 1 HEG1 Q9ULZ1 Apelin-13 APLNQ9ULZ9 Matrix metalloproteinase-17 MMP17 Q9UM21Alpha-1,3-mannosyl-glycoprotein 4-beta-N- MGAT4Aacetylglucosaminyltransferase A soluble form Q9UM22 Mammalianependymin-related protein 1 EPDR1 Q9UM73 ALK tyrosine kinase receptorALK Q9UMD9 97 kDa linear IgA disease antigen COL17A1 Q9UMX5 NeudesinNENF Q9UN73 Protocadherin alpha-6 PCDHA6 Q9UNA0 A disintegrin andmetalloproteinase with ADAMTS5 thrombospondin motifs 5 Q9UNI1Chymotrypsin-like elastase family member 1 CELA1 Q9UNK4 Group IIDsecretory phospholipase A2 PLA2G2D Q9UP79 A disintegrin andmetalloproteinase with ADAMTS8 thrombospondin motifs 8 Q9UPZ6Thrombospondin type-1 domain-containing THSD7A protein 7A Q9UQ72Pregnancy-specific beta-1-glycoprotein 11 PSG11 Q9UQ74Pregnancy-specific beta-1-glycoprotein 8 PSG8 Q9UQC9 Calcium-activatedchloride channel regulator 2 CLCA2 Q9UQE7 Structural maintenance ofchromosomes protein 3 SMC3 Q9UQP3 Tenascin-N TNN Q9Y223UDP-N-acetylglucosamine 2-epimerase GNE Q9Y240 C-type lectin domainfamily 11 member A CLEC11A Q9Y251 Heparanase 8 kDa subunit HPSE Q9Y258C-C motif chemokine 26 CCL26 Q9Y264 Angiopoietin-4 ANGPT4 Q9Y275 Tumornecrosis factor ligand superfamily member TNFSF13B 13b, membrane formQ9Y287 BRI2 intracellular domain ITM2B Q9Y2E5 Epididymis-specificalpha-mannosidase MAN2B2 Q9Y334 von Willebrand factor Adomain-containing VWA7 protein 7 Q9Y337 Kallikrein-5 KLK5 Q9Y3B3Transmembrane emp24 domain-containing protein 7 TMED7 Q9Y3E2 BolA-likeprotein 1 BOLA1 Q9Y426 C2 domain-containing protein 2 C2CD2 Q9Y4K0 Lysyloxidase homolog 2 LOXL2 Q9Y4X3 C-C motif chemokine 27 CCL27 Q9Y5C1Angiopoietin-related protein 3 ANGPTL3 Q9Y5I2 Protocadherin alpha-10PCDHA10 Q9Y5I3 Protocadherin alpha-1 PCDHA1 Q9Y5K2 Kallikrein-4 KLK4Q9Y5L2 Hypoxia-inducible lipid droplet-associated protein HILPDA Q9Y5Q5Atrial natriuretic peptide-converting enzyme CORIN Q9Y5R2 Matrixmetalloproteinase-24 MMP24 Q9Y5U5 Tumor necrosis factor receptorsuperfamily TNFRSF18 member 18 Q9Y5W5 Wnt inhibitory factor 1 WIF1Q9Y5X9 Endothelial lipase LIPG Q9Y625 Secreted glypican-6 GPC6 Q9Y646Carboxypeptidase Q CPQ Q9Y6C2 EMILIN-1 EMILIN1 Q9Y6F9 Protein Wnt-6 WNT6Q9Y6I9 Testis-expressed sequence 264 protein TEX264 Q9Y6L7 Tolloid-likeprotein 2 TLL2 Q9Y6N3 Calcium-activated chloride channel regulatorCLCA3P family member 3 Q9Y6N6 Laminin subunit gamma-3 LAMC3 Q9Y6R7IgGFc-binding protein FCGBP Q9Y6Y9 Lymphocyte antigen 96 LY96 Q9Y6Z7Collectin-10 COLEC10

In some embodiments, the compositions and methods of the inventionprovide for the delivery of one or more mRNAs encoding one or moreadditional exemplary proteins listed in Table 2; thus, compositions ofthe invention may comprise an mRNA encoding a protein listed in Table 2(or a homolog thereof) along with other components set out herein, andmethods of the invention may comprise preparing and/or administering acomposition comprising an mRNA encoding a protein chosen from theproteins listed in Table 2 (or a homolog thereof) along with othercomponents set out herein.

TABLE 2 Additional Exemplary Proteins Uniprot ID Protein Name Gene NameA6NGW2 Putative stereocilin-like protein STRCP1 A6NIE9 Putative serineprotease 29 PRSS29P A6NJ16 Putative V-set and immunoglobulin IGHV4OR15-8domain-containing-like protein IGHV4OR15-8 A6NJS3 Putative V-set andimmunoglobulin IGHV1OR21-1 domain-containing-like protein IGHV1OR21-1A6NMY6 Putative annexin A2-like protein ANXA2P2 A8MT79 Putativezinc-alpha-2-glycoprotein-like 1 A8MWS1 Putative killer cellimmunoglobulin-like KIR3DP1 receptor like protein KIR3DP1 A8MXU0Putative beta-defensin 108A DEFB108P1 C9JUS6 Putativeadrenomedullin-5-like protein ADM5 P0C7V7 Putative signal peptidasecomplex SEC11B catalytic subunit SEC11B P0C854 Putative cat eye syndromecritical region CECR9 protein 9 Q13046 Putative pregnancy-specificbeta-1- PSG7 glycoprotein 7 Q16609 Putative apolipoprotein(a)-likeprotein 2 LPAL2 Q2TV78 Putative macrophage-stimulating protein MST1P9MSTP9 Q5JQD4 Putative peptide YY-3 PYY3 Q5R387 Putative inactive groupIIC secretory PLA2G2C phospholipase A2 Q5VSP4 Putative lipocalin 1-likeprotein 1 LCN1P1 Q5W188 Putative cystatin-9-like protein CST9LP1 CST9LP1Q6UXR4 Putative serpin A13 SERPINA13P Q86SH4 Putative testis-specificprion protein PRNT Q86YQ2 Putative latherin LATH Q8IVG9 Putative humaninpeptide MT-RNR2 Q8NHM4 Putative trypsin-6 TRY6 Q8NHW4 C-C motifchemokine 4-like CCL4L2 Q9H7L2 Putative killer cell immunoglobulin-likeKIR3DX1 receptor-like protein KIR3DX1 Q9NRI6 Putative peptide YY-2 PYY2Q9UF72 Putative TP73 antisense gene protein 1 TP73-AS1 Q9UKY3 Putativeinactive carboxylesterase 4 CES1P1

The Uniprot IDs set forth in Table 1 and Table 2 refer to the humanversions the listed proteins and the sequences of each are availablefrom the Uniprot database. Sequences of the listed proteins are alsogenerally available for various animals, including various mammals andanimals of veterinary or industrial interest. Accordingly, in someembodiments, compositions and methods of the invention provide for thedelivery of one or more mRNAs encoding one or more proteins chosen frommammalian homologs or homologs from an animal of veterinary orindustrial interest of the secreted proteins listed in Table 1 or Table2; thus, compositions of the invention may comprise an mRNA encoding aprotein chosen from mammalian homologs or homologs from an animal ofveterinary or industrial interest of a protein listed in Table 1 orTable 2 along with other components set out herein, and methods of theinvention may comprise preparing and/or administering a compositioncomprising an mRNA encoding a protein chosen from mammalian homologs orhomologs from an animal of veterinary or industrial interest of aprotein listed in Table 1 or Table 2 along with other components set outherein. In some embodiments, mammalian homologs are chosen from mouse,rat, hamster, gerbil, horse, pig, cow, llama, alpaca, mink, dog, cat,ferret, sheep, goat, or camel homologs. In some embodiments, the animalof veterinary or industrial interest is chosen from the mammals listedabove and/or chicken, duck, turkey, salmon, catfish, or tilapia.

In embodiments, the compositions and methods of the invention providefor the delivery of mRNA encoding a lysosomal protein chosen from Table3. In some embodiments, the compositions and methods of the inventionprovide for the delivery of one or more mRNAs encoding one or morelysosomal and/or related proteins listed in Table 3; thus, compositionsof the invention may comprise an mRNA encoding a protein listed in Table3 (or a homolog thereof) along with other components set out herein, andmethods of the invention may comprise preparing and/or administering acomposition comprising an mRNA encoding a protein chosen from theproteins listed in Table 3 (or a homolog thereof) along with othercomponents set out herein.

TABLE 3 Lysosomal and Related Proteins α-fucosidase α-galactosidaseα-glucosidase α-Iduronidase α-mannosidase α-N-acetylgalactosaminidase(α-galactosidase B) β-galactosidase β-glucuronidase β-hexosaminidaseβ-mannosidase 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase3-methylcrotonyl-CoA carboxylase 3-O-sulfogalactosyl cerebrosidesulfatase (arylsulfatase A) acetyl-CoA transferase acidalpha-glucosidase acid ceramidase acid lipase acid phosphatase acidsphingomyelinase alpha-galactosidase A arylsulfatase Abeta-galactosidase beta-glucocerebrosidase beta-hexosaminidasebiotinidase cathepsin A cathepsin K CLN3 CLN5 CLN6 CLN8 CLN9 cystinetransporter (cystinosin) cytosolic protein beta3A subunit of the adaptorprotein-3 complex, AP3 formyl-Glycine generating enzyme (FGE)galactocerebrosidase galactose-1-phosphate uridyltransferase (GALT)galactose 6-sulfate sulfatase (also known asN-acetylgalactosamine-6-sulfatase) glucocerebrosidase glucuronatesulfatase glucuronidase glycoprotein cleaving enzymes glycosaminoglycancleaving enzymes glycosylasparaginase (aspartylglucosaminidase) GM2-APHeparan-alpha-glucosaminide N-acetyltransferase (HGSNAT, TMEM76) Heparansulfatase hexosaminidase A lysosomal proteases methylmalonyl-CoA mutasehyaluronidase Iduronate sulfatase LAMP-2 lysosomal α-mannosidaseLysosomal p40 (C2orf18) Major facilitator superfamily domain containing8 protein (MFSD8 or CLN7) N-acetylgalactosamine 4-sulfatase N-acetylglucosamine 6-sulfatase N-acetyl glucosaminidaseN-acetylglucosamine-1-phosphate transferase NPC1 NPC2 palmitoyl-proteinthioesterase palmitoyl-protein thioesterase (CLN1) Saposin A(Sphingolipid activator protein A) Saposin B (Sphingolipid activatorprotein B) Saposin C (Sphingolipid activator protein C) Saposin D(Sphingolipid activator protein D) sialic acid transporter (sialin)sialidase Sialin sulfatase Transmembrane protein 74 (TMEM74)tripeptidyl-peptidase tripeptidyl-peptidase I (CLN2)UDP-N-acetylglucosamine-phosphotransferase

Information regarding lysosomal proteins is available from Lubke et al.,“Proteomics of the Lysosome,” Biochim Biophys Acta. (2009) 1793:625-635. In some embodiments, the protein listed in Table 3 and encodedby mRNA in the compositions and methods of the invention is a humanprotein. Sequences of the listed proteins are also available for variousanimals, including various mammals and animals of veterinary orindustrial interest as described above.

In some embodiments, the compositions and methods of the inventionprovide for the delivery of mRNA encoding a therapeutic protein (e.g.,cytosolic, transmembrane or secreted) such as those listed in Table 4.In some embodiments, the compositions and methods of the inventionprovide for the delivery of an mRNA encoding a therapeutic proteinuseful in treating a disease or disorder (i.e., indication) listed inTable 4; thus, compositions of the invention may comprise an mRNAencoding a therapeutic protein listed or not listed in Table 4 (or ahomolog thereof, as discussed below) along with other components set outherein for treating a disease or disorder (i.e., indication) listed inTable 4, and methods of the invention may comprise preparing and/oradministering a composition comprising an mRNA encoding a such a protein(or a homolog thereof, as discussed below) along with other componentsset out herein for treatment of a disease or disorder listed in Table 4.

TABLE 4 Exemplary Indications and Related Proteins IndicationTherapeutic Protein 3-Methylcrotonyl-CoA carboxylase deficiencyMethylcrotonoyl-CoA carboxylase 3-Methylglutaconic aciduriaMethylglutaconyl-CoA hydratase Actinic keratosis Acute intermittentporphyria Porphobilinogen deaminase Acute lymphocytic leukemia Acutemyeloid leukemia Addison's disease Adenosine deaminase deficiencyAdenosine deaminase Adrenoleukodystrophy ABCD1 AdrenomyeloneuropathyAIDS/HIV Alcohol use disorders Alkaptonuria Homogentisate1,2-dioxygenase Allergic asthma Anti-IgE mAb Allergies (dermatitis,rhinitis) Alopecia areata Alpers' disease POLG Alpers-Huttenlochersyndrome Alpha 1-antitrypsin deficiency Alpha 1 protease inhibitorAlpha-mannosidosis Alpha-D-mannosidase Alport syndrome Alzheimer'sdisease Amyloid light-chain amyloidosis Amyotrophic lateral sclerosis(ALS) Anemia Erythropoietin Aortic valve stenosis Argininemia ArginaseArgininosuccinic acidemia Argininosuccinate lyase Arrhythmogenic rightventricular dysplasia Autism Autosomal dominant and recessiveprogressive external ophthalmoplegia with mitochondrial DNA deletionsAutosomal recessive polycystic kidney disease ARPKD Bacterial infectionsBasal cell carcinoma Batten disease Battenin + others B-cell chroniclymphocytic leukemia Becker muscular dystrophy DystrophinBeta-thalassemia Beta globin Binge eating disorder Bipolar disorderBladder cancer Blepharospasm, Cervical dystonia, Chronic migraine,Botulinum toxin more Bronchiolitis obliterans Brugada syndrome Buerger'sdisease CACNA1A CACNB4-related Episodic Ataxia Type 2 Cancer anddepression Cancer and sexual dysfunction Cancer in pregnancyCarbamylphosphate synthetase deficiency Carbamylphosphate synthetaseCarcinoma of the gallbladder Cardiomyopathy (diabetic) Cardiomyopathy(hypertrophic) Carnitine uptake defect SLC22A5 Catecholaminergicpolymorphic ventricular tachycardia CDKL5-related Atypical Rett SyndromeCeliac disease Cellulitis Cerebrovascular disease Cervix uteri cancerChronic fatigue syndrome Chronic graft versus host disease Chronicidiopathic urticaria Chronic immune thrombocytopenia ThrombopoietinChronic kidney kisease Chronic liver disease Chronic lymphocyticleukemia Chronic myeloid leukemia Chronic pancreatitis Cirrhosis of theliver Citrullinemia, type I Argininosuccinate synthase Classic RettSyndrome Classical galactosemia Galactose-1-phosphateuridylyltransferase Clostridium difficile associated diarrhea Clottingdisorders COAD/COPD Cocaine addiction COL4A5-related disorders Coldcontact urticaria Contraception, female Coronary artery diseases Corpusuteri cancer Corticobasal degeneration Crigler-Najjar syndromeUDP-glucuronosyltransferase Critical limb ischemia CTNS-relatedcystinosis Cutaneous lupus erythematosus Cutaneous neuroendocrinecarcinoma (Merkel Cell) Cystic fibrosis CFTR Cystic fibrosisDeoxyribonuclease I Cystinosis Cystinosin Cystinuria SLC7A9 Dementia(Lewy body) Depression Diabetic foot infections Diabetic foot ulcerDiabetic peripheral neuropathy Diabetic ulcers Diarrhoeal diseasesDiffuse large B-cell lymphoma DiGeorge syndrome Diverticulitis Drug usedisorders Duchenne muscular dystrophy Dystrophin Dysarthria Dyskinesia(levodopa-induced) Early-onset autosomal dominant Alzheimer's diseaseEczema Ehlers-Danlos syndrome, type 1 EIF2B1 EIF2B2 EIF2B3 EIF2B4EIF2B5-related childhood ataxia with central nervous systemhypomyelination/vanishing white matter Eosinophilic esophagitis EpilepsyErectile dysfunction Erythropoietic protoporphyria FerrochelataseEsophageal carcinoma Essential tremor Fabry disease Alpha galactosidaseFamilial adenomatous polyposis APC Familial chylomicronemia Lipoproteinlipase Familial dysbetalipoproteinemia Apolipoprotein E Familialisolated dilated cardiomyopathy Familial mediterranean fever Pyrin(MEFV) Familial melanoma Female infertility Follicle stimulating hormoneFemale sexual dysfunction Fibromyalgia FMR1-related disorders Fracturehealing Fragile X Premature Ovarian Failure Syndrome Fragile X syndromeFMRP Fragile X-Associated Tremor/Ataxia Syndrome Friedreich's ataxiaFrontotemporal dementia Fryns syndrome Galactocerebrosidase deficienciesGALE deficiency Galactose epimerase GALK deficiency GalactokinaseGALT-related galactosemia Gastric cancer Gastroesophageal reflux diseaseGaucher disease Glucocerebrosidase Gilbert syndromeUDP-glucuronosyltransferase Glioblastoma multiforme GlomerulonephritisGlutaric acidemia, type I Glutaryl-CoA dehydrogenase GM2 gangliosidosisHEXA, HEXB Gout Urate oxidase Graft versus host disease Growth hormonedeficiency Growth hormone 1/Growth hormone 2 Head and neck cancer,Metastatic colorectal cancer Anti-EGFr mAb Hearing loss, adult onsetHeart failure Hemachromatosis HFE protein Hemifacial spasm Hemolyticuremic syndrome Anti-complement factor C5 mAb Hemophilia A Factor VIIIHemophilia A, Hemophilia B Factor VII Hemophilia B Factor IX HepatitisB, Hepatitis C Interferon alpha HER2+ breast cancer, gastric cancerAnti-HER2 mAb Hereditary angioedema C1 esterase inhibitor Hereditaryhemorrhagic telangiectasia Hereditary hemorrhagic telangiectasia (AT)Hereditary spherocytosis Hidradenitis suppurativa HomocystinuriaCystathionine beta-synthase Homozygous familial hypercholesterolemia LDLreceptor Hunter syndrome (MPS II) Iduronate-2-sulfatase Huntingtondisease Huntingtin Hurler syndrome (MPS I) Alpha-L iduronidaseHydrolethalus Hyperalgesia Hyperbilirubinemia HyperhidrosisHyperlipidemia Hypermethioninemia Methionine adenosyltransferaseHyperoxaluria, type I Serine-pyruvate aminotransferase HypertensionHyperuricemia Hyponatremia Hypoparathyroidism Parathyroid hormoneHypophosphatasia TNSALP Idiopathic pulmonary fibrosis IminoglycinuriaImmunoglobulin deficiency Immunoglobulin Infection (adenovirus)Infection (anthrax prophylaxis) Infection (BK virus) Infection(Clostridium difficile prophylaxis) Infection (Dengue fever prophylaxis)Infection (Epstein-Barr virus) Infection (Hepatitis-D) Infection (Lymedisease prophylaxis) Infection (Smallpox virus) Infectious diseasesvaccines Infectious antigen Inflammatory heart diseases InsomniaInterstitial cystitis Iron-deficiency anaemia Irritable bowel diseaseIschaemic heart disease Isovaleric aciduria Isovaleric acid CoAdehydrogenase deficiency Jansky-Bielschowsky disease Juvenile Battendisease Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) Juvenilerheumatoid arthritis TNF-alpha inhibitors Kennedy's disease (SBMA)Keratoconus Krabbe disease Galactocerebrosidase Leber's hereditary opticneuropathy NADH dehydrogenase Leiomyosarcoma Lennox-Gastaut syndromeLesch-Nyhan syndrome Hypoxanthine phosphoribosyltransferase 1 LeukaemiaLi-Fraumeni syndrome TP53 Lipoma Liposarcoma Liver cancer Long-chain3-OH acyl-CoA dehydrogenase deficiency Long-chain-3-hydroxyacyl-CoAdehydrogenase Lower respiratory infections Lysosomal acid lipasedeficiency Lysosomal acid lipase Macular degeneration Major depressivedisorder Malignant fibrous histiocytoma Mantle cell lymphoma Maple syrupurine disease 3-methyl-2-oxobutanoate dehydrogenase Marfan syndrome FBN1Maroteaux-Lamy syndrome (MPS VI) N-acetylgalactosamine 4-sulfataseMastocytosis McArdle disease Muscle glycogen phosphorylase MECP2-relateddisorders MECP2-related Severe Neonatal Encephalopathy Medium-chainacyl-CoA dehydrogenase deficiency Acyl-CoA dehydrogenase MelanomaAnti-CTLA4 mAb Metachromatic leukodystrophy Arylsulfatase A Metastaticcolorectal cancer, NSCLC, others Anti-VEGF mAb Methylmalonyl-CoA mutasedeficiency Methylmalonyl-CoA mutase Migraine Mitochondrial oxidativephosphorylation disorders Morquio syndrome, type A (MPS IVA) Galactose6-sulfate sulfatase Morquio syndrome, type B (MPS IVB)Beta-galactosidase Mouth and oropharynx cancers Multiple carboxylasedeficiency Biotin-methylcrotonoyl-CoA-carboxylase ligase Multiplemyeloma Multiple sclerosis Anti-VLA-4 mAb Multiple sclerosis Interferonbeta Multiple system atrophy Myasthenia gravis Myelofibrosis NarcolepsyNeonatal bronchopulmonary dysplasia Neonatal infections Nephritis andnephrosis Neurofibromatosis, type 1 NF-1 Neuronal ceroidlipofuscinoses-related diseases Neutropenia G-CSF Niemann Pick disease,type A/B SMPD1 Niemann Pick disease, type C NPC1 Niemann-Pick diseaseType C1 Nocturia Non-alcoholic fatty liver disease Non-Hodgkin lymphomaAnti-CD20 mAb Non-small cell lung cancer Notch-3 related cerebralautosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL) Obesity Ophthalmoparesis Opioid inducedconstipation Ornithine transcarbamylase deficiency Ornithinetranscarbamylase Osteoarthritis Osteopetrosis Osteoporosis Anti-RANKLmAb Ovarian cancer Paget disease of bone Sequestosome 1 Pain Pancreaticcarcinoma Panic disorder Parkinson disease Paroxysmal nocturnalhemoglobinuria Anti-complement factor C5 Mab Pediculosis capitis (headlice) Pelizaeus-Merzbacher disease Pemphigus vulgaris Peptic ulcerdisease Peripheral neuropathy Peyronie's disease PhenylketonuriaPhenylalanine hydroxylase Pneumococcal infection prophylaxisPOLG-related sensory ataxic neuropathy Polycystic kidney diseasePolycystic ovary syndrome Polycythaemia vera Polymerase G-relateddisorders Polymorphous light eruption Pompe disease Alpha glucosidasePorphyria cutanea tarda Uroporphyrinogen decarboxylase Post herpeticneuralgia Post-organ transplant Pouchitis PPM-X Syndrome Prader-Willisyndrome Preeclampsia Premature ejaculation Prematurity and low birthweight Primary ciliary dyskinesia Primary glomerular diseases Primaryhumoral immune deficiencies (e.g., CVID) Immunoglobulin ProctitisProgressive multifocal leukoencephalopathy Progressive supranuclearpalsy Propionic acidemia Propionyl-CoA carboxylase Prostate cancerPsoriasis Anti-IL-12 & IL-23 mAb Psoriatic arthritis TNF-alphainhibitors PTT-1 Pulmonary arterial hypertension Pulmonary arterialhypertension Raynaud's phenomenon Refractive errors Renal cell carcinomaRestless leg syndrome Retinitis pigmentosa Rheumatic heart diseaseRheumatoid arthritis Anti-interleukin-6 (IL-6) mAb Rheumatoid arthritisT-cell costimulation blocker Rheumatoid arthritis TNF-alpha inhibitorRomano-Ward syndrome Rosacea Sanfilippo syndrome, type A (MPS IIIA)Heparan N-sulfatase Sanfilippo syndrome, type B (MPS IIIB)N-acetyl-alpha-D-glucosaminidase Santavuori-Haltia disease SchizophreniaSchnitzler syndrome Scleroderma SCN1A SCN1B-related seizure disordersShort-chain acyl-CoA dehydrogenase deficiency Butyryl-CoA dehydrogenaseSickle cell disease Hemoglobin SLC3A1-related disorders Small cell lungcancer SMN-1-related spinal muscular atrophy (SMA) Spinal muscularatrophy Survival motor neuron protein Squamous cell carcinoma of headand neck Stickler syndrome Stomach cancer Stroke prophylaxis Synovialsarcoma Systemic lupus erythematosus Anti-BAFF Systemic sclerosisTetrahydrobiopterin-deficient hyperphenylalaninemia TetrahydrobiopterinThromboangiitis obliterans Thrombotic disorders Thyroid cancer TPP1deficiencies Trachea, bronchus, lung cancers Tricuspid atresia TSC1TSC2-related tuberous sclerosis Type 2 diabetes mellitus Glucagon-likepeptide 1 (GLP-1) agonist Type 2 diabetes mellitus Insulin Tyrosinemia,type I Fumarylacetoacetase Ulcerative colitis Uterine fibroids Varicoseveins Venous thromboembolism Very long-chain acyl-CoA dehydrogenasedeficiency Long-chain-acyl-CoA dehydrogenase von Gierke's diseaseGlucose-6-phosphatase Von Hippel-Lindau disease pVHL Wegenergranulomatosis Wilson disease Wilson disease protein X-Linked adrenalhypoplasia X-linked adrenoleukodystrophy X-linked agammaglobulinemiaBruton's tyrosine kinase

In some embodiments, the present invention is used to prevent, treatand/or cure a subject affected with a disease or disorder listed orassociated with the proteins listed in Tables 1, 2, 3 or 4. In someembodiments, an mRNA encodes one or more of argininosuccinate synthetase(ASS1), Factor IX, survival motor neuron 1 (SMN1), or phenylalaninehydroxylase

Synthesis of mRNA

mRNAs according to the present invention may be synthesized according toany of a variety of known methods. For example, mRNAs according to thepresent invention may be synthesized via in vitro transcription (IVT).Briefly, IVT is typically performed with a linear or circular DNAtemplate containing a promoter, a pool of ribonucleotide triphosphates,a buffer system that may include DTT and magnesium ions, and anappropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAseI, pyrophosphatase, and/or RNAse inhibitor. The exact conditions willvary according to the specific application.

In some embodiments, for the preparation of mRNA according to theinvention, a DNA template is transcribed in vitro. A suitable DNAtemplate typically has a promoter, for example a T3, T7 or SP6 promoter,for in vitro transcription, followed by desired nucleotide sequence fordesired mRNA and a termination signal.

Desired mRNA sequence(s) according to the invention may be determinedand incorporated into a DNA template using standard methods. Forexample, starting from a desired amino acid sequence (e.g., an enzymesequence), a virtual reverse translation is carried out based on thedegenerated genetic code. Optimization algorithms may then be used forselection of suitable codons. Typically, the G/C content can beoptimized to achieve the highest possible G/C content on one hand,taking into the best possible account the frequency of the tRNAsaccording to codon usage on the other hand. The optimized RNA sequencecan be established and displayed, for example, with the aid of anappropriate display device and compared with the original (wild-type)sequence. A secondary structure can also be analyzed to calculatestabilizing and destabilizing properties or, respectively, regions ofthe RNA.

Modified mRNA

In some embodiments, mRNA according to the present invention may besynthesized as unmodified or modified mRNA. Typically, mRNAs aremodified to enhance stability. Modifications of mRNA can include, forexample, modifications of the nucleotides of the RNA. An modified mRNAaccording to the invention can thus include, for example, backbonemodifications, sugar modifications or base modifications. In someembodiments, mRNAs may be synthesized from naturally occurringnucleotides and/or nucleotide analogues (modified nucleotides)including, but not limited to, purines (adenine (A), guanine (G)) orpyrimidines (thymine (T), cytosine (C), uracil (U)), and as modifiednucleotides analogues or derivatives of purines and pyrimidines, such ase.g. 1-methyl-adenine, 2-methyl-adenine,2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine,N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine,4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine,1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine,7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil),dihydro-uracil, 2-thio-uracil, 4-thio-uracil,5-carboxymethylaminomethyl-2-thio-uracil,5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil,5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil,5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester,5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil,5′-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyaceticacid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil,queosine, .beta.-D-mannosyl-queosine, wybutoxosine, andphosphoramidates, phosphorothioates, peptide nucleotides,methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine. Thepreparation of such analogues is known to a person skilled in the arte.g. from the U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S.Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707,U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No.5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No. 5,153,319, U.S. Pat.Nos. 5,262,530 and 5,700,642, the disclosures of which are incorporatedby reference in their entirety.

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may contain RNAbackbone modifications. Typically, a backbone modification is amodification in which the phosphates of the backbone of the nucleotidescontained in the RNA are modified chemically. Exemplary backbonemodifications typically include, but are not limited to, modificationsfrom the group consisting of methylphosphonates, methylphosphoramidates,phosphoramidates, phosphorothioates (e.g. cytidine5′-O-(1-thiophosphate)), boranophosphates, positively chargedguanidinium groups etc., which means by replacing the phosphodiesterlinkage by other anionic, cationic or neutral groups.

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may containsugar modifications. A typical sugar modification is a chemicalmodification of the sugar of the nucleotides it contains including, butnot limited to, sugar modifications chosen from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide (2′-fluoro-2′-deoxycytidine5′-triphosphate, 2′-fluoro-2′-deoxyuridine 5′-triphosphate),2′-deoxy-2′-deamine-oligoribonucleotide (2′-amino-2′-deoxycytidine5′-triphosphate, 2′-amino-2′-deoxyuridine 5′-triphosphate),2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyloligoribonucleotide(2′-O-methylcytidine 5′-triphosphate, 2′-methyluridine 5′-triphosphate),2′-C-alkyloligoribonucleotide, and isomers thereof (2′-aracytidine5′-triphosphate, 2′-arauridine 5′-triphosphate), or azidotriphosphates(2′-azido-2′-deoxycytidine 5′-triphosphate, 2′-azido-2′-deoxyuridine5′-triphosphate).

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) may containmodifications of the bases of the nucleotides (base modifications). Amodified nucleotide which contains a base modification is also called abase-modified nucleotide. Examples of such base-modified nucleotidesinclude, but are not limited to, 2-amino-6-chloropurine riboside5′-triphosphate, 2-aminoadenosine 5′-triphosphate, 2-thiocytidine5′-triphosphate, 2-thiouridine 5′-triphosphate, 4-thiouridine5′-triphosphate, 5-aminoallylcytidine 5′-triphosphate,5-aminoallyluridine 5′-triphosphate, 5-bromocytidine 5′-triphosphate,5-bromouridine 5′-triphosphate, 5-iodocytidine 5′-triphosphate,5-iodouridine 5′-triphosphate, 5-methylcytidine 5′-triphosphate,5-methyluridine 5′-triphosphate, 6-azacytidine 5′-triphosphate,6-azauridine 5′-triphosphate, 6-chloropurine riboside 5′-triphosphate,7-deazaadenosine 5′-triphosphate, 7-deazaguanosine 5′-triphosphate,8-azaadenosine 5′-triphosphate, 8-azidoadenosine 5′-triphosphate,benzimidazole riboside 5′-triphosphate, N1-methyladenosine5′-triphosphate, N1-methylguanosine 5′-triphosphate, N6-methyladenosine5′-triphosphate, 06-methylguanosine 5′-triphosphate, pseudouridine5′-triphosphate, puromycin 5′-triphosphate or xanthosine5′-triphosphate.

Cap Structure

Typically, mRNA synthesis includes the addition of a “cap” on theN-terminal (5′) end, and a “tail” on the C-terminal (3′) end. Thepresence of the cap is important in providing resistance to nucleasesfound in most eukaryotic cells. The presence of a “tail” serves toprotect the mRNA from exonuclease degradation.

Thus, in some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a5′ cap structure. A 5′ cap is typically added as follows: first, an RNAterminal phosphatase removes one of the terminal phosphate groups fromthe 5′ nucleotide, leaving two terminal phosphates; guanosinetriphosphate (GTP) is then added to the terminal phosphates via aguanylyl transferase, producing a 5′5′5 triphosphate linkage; and the7-nitrogen of guanine is then methylated by a methyltransferase.Examples of cap structures include, but are not limited to, m7G(5′)ppp(5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

In some embodiments, naturally occurring cap structures comprise a7-methyl guanosine that is linked via a triphosphate bridge to the5′-end of the first transcribed nucleotide, resulting in a dinucleotidecap of m⁷G(5′)ppp(5′)N, where N is any nucleoside. In vivo, the cap isadded enzymatically. The cap is added in the nucleus and is catalyzed bythe enzyme guanylyl transferase. The addition of the cap to the 5′terminal end of RNA occurs immediately after initiation oftranscription. The terminal nucleoside is typically a guanosine, and isin the reverse orientation to all the other nucleotides, i.e.,G(5′)ppp(5′)GpNpNp.

A common cap for mRNA produced by in vitro transcription ism⁷G(5′)ppp(5′)G, which has been used as the dinucleotide cap intranscription with T7 or SP6 RNA polymerase in vitro to obtain RNAshaving a cap structure in their 5′-termini. The prevailing method forthe in vitro synthesis of capped mRNA employs a pre-formed dinucleotideof the form m⁷G(5′)ppp(5′)G (“m⁷GpppG”) as an initiator oftranscription.

To date, a usual form of a synthetic dinucleotide cap used in in vitrotranslation experiments is the Anti-Reverse Cap Analog (“ARCA”) ormodified ARCA, which is generally a modified cap analog in which the 2′or 3′ OH group is replaced with —OCH₃.

Additional cap analogs include, but are not limited to, chemicalstructures selected from the group consisting of m⁷GpppG, m⁷GpppA,m⁷GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analog(e.g., m^(2,7)GpppG), trimethylated cap analog (e.g., m^(2,2,7)GpppG),dimethylated symmetrical cap analogs (e.g., m⁷Gpppm⁷G), or anti reversecap analogs (e.g., ARCA; m^(7,2′Ome)GpppG, m^(72′d)GpppG,m^(7,3′Ome)GpppG, m^(7,3′d)GpppG and their tetraphosphate derivatives)(see, e.g., Jemielity, J. et al., “Novel ‘anti-reverse’ cap analogs withsuperior translational properties”, RNA, 9: 1108-1122 (2003)).

In some embodiments, a suitable cap is a 7-methyl guanylate (“m⁷G”)linked via a triphosphate bridge to the 5′-end of the first transcribednucleotide, resulting in m⁷G(5′)ppp(5′)N, where N is any nucleoside. Apreferred embodiment of a m⁷G cap utilized in embodiments of theinvention is m⁷G(5′)ppp(5′)G.

In some embodiments, the cap is a Cap0 structure. Cap0 structures lack a2′-O-methyl residue of the ribose attached to bases 1 and 2. In someembodiments, the cap is a Cap1 structure. Cap1 structures have a2′-O-methyl residue at base 2. In some embodiments, the cap is a Cap2structure. Cap2 structures have a 2′-O-methyl residue attached to bothbases 2 and 3.

A variety of m⁷G cap analogs are known in the art, many of which arecommercially available. These include the m⁷GpppG described above, aswell as the ARCA 3′-OCH₃ and 2′-OCH₃ cap analogs (Jemielity, J. et al.,RNA, 9: 1108-1122 (2003)). Additional cap analogs for use in embodimentsof the invention include N7-benzylated dinucleoside tetraphosphateanalogs (described in Grudzien, E. et al., RNA, 10: 1479-1487 (2004)),phosphorothioate cap analogs (described in Grudzien-Nogalska, E., etal., RNA, 13: 1745-1755 (2007)), and cap analogs (including biotinylatedcap analogs) described in U.S. Pat. Nos. 8,093,367 and 8,304,529,incorporated by reference herein.

Tail Structure

Typically, the presence of a “tail” serves to protect the mRNA fromexonuclease degradation. The poly A tail is thought to stabilize naturalmessengers and synthetic sense RNA. Therefore, in certain embodiments along poly A tail can be added to an mRNA molecule thus rendering the RNAmore stable. Poly A tails can be added using a variety of art-recognizedtechniques. For example, long poly A tails can be added to synthetic orin vitro transcribed RNA using poly A polymerase (Yokoe, et al. NatureBiotechnology. 1996; 14: 1252-1256). A transcription vector can alsoencode long poly A tails. In addition, poly A tails can be added bytranscription directly from PCR products. Poly A may also be ligated tothe 3′ end of a sense RNA with RNA ligase (see, e.g., Molecular CloningA Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis(Cold Spring Harbor Laboratory Press: 1991 edition)).

In some embodiments, mRNAs (e.g., enzyme encoding mRNAs) include a 3′poly(A) tail structure. Typically, the length of the poly A tail can beat least about 10, 50, 100, 200, 300, 400 at least 500 nucleotides (SEQID NO: 12). In some embodiments, a poly-A tail on the 3′ terminus ofmRNA typically includes about 10 to 300 adenosine nucleotides (SEQ IDNO: 13) (e.g., about 10 to 200 adenosine nucleotides, about 10 to 150adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).In some embodiments, mRNAs include a 3′ poly(C) tail structure. Asuitable poly-C tail on the 3′ terminus of mRNA typically include about10 to 200 cytosine nucleotides (SEQ ID NO: 14) (e.g., about 10 to 150cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about10 to 40 cytosine nucleotides). The poly-C tail may be added to thepoly-A tail or may substitute the poly-A tail.

In some embodiments, the length of the poly A or poly C tail is adjustedto control the stability of a modified sense mRNA molecule of theinvention and, thus, the transcription of protein. For example, sincethe length of the poly A tail can influence the half-life of a sensemRNA molecule, the length of the poly A tail can be adjusted to modifythe level of resistance of the mRNA to nucleases and thereby control thetime course of polynucleotide expression and/or polypeptide productionin a target cell.

5′ and 3′ Untranslated Region

In some embodiments, mRNAs include a 5′ and/or 3′ untranslated region.In some embodiments, a 5′ untranslated region includes one or moreelements that affect an mRNA's stability or translation, for example, aniron responsive element. In some embodiments, a 5′ untranslated regionmay be between about 50 and 500 nucleotides in length.

In some embodiments, a 3′ untranslated region includes one or more of apolyadenylation signal, a binding site for proteins that affect anmRNA's stability of location in a cell, or one or more binding sites formiRNAs. In some embodiments, a 3′ untranslated region may be between 50and 500 nucleotides in length or longer.

Exemplary 3′ and/or 5′ UTR sequences can be derived from mRNA moleculeswhich are stable (e.g., globin, actin, GAPDH, tubulin, histone, orcitric acid cycle enzymes) to increase the stability of the sense mRNAmolecule. For example, a 5′ UTR sequence may include a partial sequenceof a CMV immediate-early 1 (IE1) gene, or a fragment thereof to improvethe nuclease resistance and/or improve the half-life of thepolynucleotide. Also contemplated is the inclusion of a sequenceencoding human growth hormone (hGH), or a fragment thereof to the 3′ endor untranslated region of the polynucleotide (e.g., mRNA) to furtherstabilize the polynucleotide. Generally, these modifications improve thestability and/or pharmacokinetic properties (e.g., half-life) of thepolynucleotide relative to their unmodified counterparts, and include,for example modifications made to improve such polynucleotides'resistance to in vivo nuclease digestion.

According to various embodiments, any size mRNA may be encapsulated byprovided liposomes. In some embodiments, the provided liposomes mayencapsulate mRNA of greater than about 0.5 kb, 1 kb, 1.5 kb, 2 kb, 2.5kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.

Formation of Liposomes

The liposomes for use in provided compositions can be prepared byvarious techniques which are presently known in the art. For example,multilamellar vesicles (MLV) may be prepared according to conventionaltechniques, such as by depositing a selected lipid on the inside wall ofa suitable container or vessel by dissolving the lipid in an appropriatesolvent, and then evaporating the solvent to leave a thin film on theinside of the vessel or by spray drying. An aqueous phase may then addedto the vessel with a vortexing motion which results in the formation ofMLVs. Uni-lamellar vesicles (ULV) can then be formed by homogenization,sonication or extrusion of the multi-lamellar vesicles. In addition,unilamellar vesicles can be formed by detergent removal techniques.

In certain embodiments, provided compositions comprise a liposomewherein the mRNA is associated on both the surface of the liposome andencapsulated within the same liposome. For example, during preparationof the compositions of the present invention, cationic liposomes mayassociate with the mRNA through electrostatic interactions. For example,during preparation of the compositions of the present invention,cationic liposomes may associate with the mRNA through electrostaticinteractions.

In some embodiments, the compositions and methods of the inventioncomprise mRNA encapsulated in a liposome. In some embodiments, the oneor more mRNA species may be encapsulated in the same liposome. In someembodiments, the one or more mRNA species may be encapsulated indifferent liposomes. In some embodiments, the mRNA is encapsulated inone or more liposomes, which differ in their lipid composition, molarratio of lipid components, size, charge (Zeta potential), targetingligands and/or combinations thereof. In some embodiments, the one ormore liposome may have a different composition of cationic lipids,neutral lipid, PEG-modified lipid and/or combinations thereof. In someembodiments the one or more lipisomes may have a different molar ratioof cationic lipid, neutral lipid, cholesterol and PEG-modified lipidused to create the liposome.

The process of incorporation of a desired mRNA into a liposome is oftenreferred to as “loading”. Exemplary methods are described in Lasic, etal., FEBS Lett., 312: 255-258, 1992, which is incorporated herein byreference. The liposome-incorporated nucleic acids may be completely orpartially located in the interior space of the liposome, within thebilayer membrane of the liposome, or associated with the exteriorsurface of the liposome membrane. The incorporation of a nucleic acidinto liposomes is also referred to herein as “encapsulation” wherein thenucleic acid is entirely contained within the interior space of theliposome. The purpose of incorporating a mRNA into a transfer vehicle,such as a liposome, is often to protect the nucleic acid from anenvironment which may contain enzymes or chemicals that degrade nucleicacids and/or systems or receptors that cause the rapid excretion of thenucleic acids. Accordingly, in some embodiments, a suitable deliveryvehicle is capable of enhancing the stability of the mRNA containedtherein and/or facilitate the delivery of mRNA to the target cell ortissue.

Liposome Size

Suitable liposomes in accordance with the present invention may be madein various sizes. In some embodiments, provided liposomes may be madesmaller than previously known mRNA encapsulating liposomes. In someembodiments, decreased size of liposomes is associated with moreefficient delivery of mRNA. Selection of an appropriate liposome sizemay take into consideration the site of the target cell or tissue and tosome extent the application for which the liposome is being made.

In some embodiments, an appropriate size of liposome is selected tofacilitate systemic distribution of antibody encoded by the mRNA. Insome embodiments, it may be desirable to limit transfection of the mRNAto certain cells or tissues. For example, to target hepatocytes aliposome may be sized such that its dimensions are smaller than thefenestrations of the endothelial layer lining hepatic sinusoids in theliver; in such cases the liposome could readily penetrate suchendothelial fenestrations to reach the target hepatocytes.

Alternatively or additionally, a liposome may be sized such that thedimensions of the liposome are of a sufficient diameter to limit orexpressly avoid distribution into certain cells or tissues. For example,a liposome may be sized such that its dimensions are larger than thefenestrations of the endothelial layer lining hepatic sinusoids tothereby limit distribution of the liposomes to hepatocytes.

In some embodiments, the size of a liposome is determined by the lengthof the largest diameter of the liposome particle. In some embodiments, asuitable liposome has a size of or less than about 500 nm, 450 nm, 400nm, 350 nm, 300 nm, 250 nm, 200 nm, 150 nm, 125 nm, 110 nm, 100 nm, 95nm, 90 nm, 85 nm, 80 nm, 75 nm, 70 nm, 65 nm, 60 nm, 55 nm, or 50 nm. Insome embodiments, a suitable liposome has a size no greater than about250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125nm, 100 nm, 75 nm, or 50 nm). In some embodiments, a suitable liposomehas a size ranging from about 10-250 nm (e.g., ranging from about 10-225nm, 10-200 nm, 10-175 nm, 10-150 nm, 10-125 nm, 10-100 nm, 10-75 nm, or10-50 nm). In some embodiments, a suitable liposome has a size rangingfrom about 100-250 nm (e.g., ranging from about 100-225 nm, 100-200 nm,100-175 nm, 100-150 nm). In some embodiments, a suitable liposome has asize ranging from about 10-100 nm (e.g., ranging from about 10-90 nm,10-80 nm, 10-70 nm, 10-60 nm, or 10-50 nm).

A variety of alternative methods known in the art are available forsizing of a population of liposomes. One such sizing method is describedin U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicatinga liposome suspension either by bath or probe sonication produces aprogressive size reduction down to small ULV less than about 0.05microns in diameter. Homogenization is another method that relies onshearing energy to fragment large liposomes into smaller ones. In atypical homogenization procedure, MLV are recirculated through astandard emulsion homogenizer until selected liposome sizes, typicallybetween about 0.1 and 0.5 microns, are observed. The size of theliposomes may be determined by quasi-electric light scattering (QELS) asdescribed in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-150 (1981),incorporated herein by reference. Average liposome diameter may bereduced by sonication of formed liposomes. Intermittent sonicationcycles may be alternated with QELS assessment to guide efficientliposome synthesis.

Pharmaceutical Compositions

To facilitate expression of mRNA in vivo, delivery vehicles such asliposomes can be formulated in combination with one or more additionalnucleic acids, carriers, targeting ligands or stabilizing reagents, orin pharmacological compositions where it is mixed with suitableexcipients. Techniques for formulation and administration of drugs maybe found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co.,Easton, Pa., latest edition.

Provided liposomally-encapsulated or associated mRNAs, and compositionscontaining the same, may be administered and dosed in accordance withcurrent medical practice, taking into account the clinical condition ofthe subject, the site and method of administration, the scheduling ofadministration, the subject's age, sex, body weight and other factorsrelevant to clinicians of ordinary skill in the art. The “effectiveamount” for the purposes herein may be determined by such relevantconsiderations as are known to those of ordinary skill in experimentalclinical research, pharmacological, clinical and medical arts. In someembodiments, the amount administered is effective to achieve at leastsome stabilization, improvement or elimination of symptoms and otherindicators as are selected as appropriate measures of disease progress,regression or improvement by those of skill in the art. For example, asuitable amount and dosing regimen is one that causes at least transientprotein (e.g., enzyme) production.

Suitable routes of administration include, for example, oral, rectal,vaginal, transmucosal, pulmonary including intratracheal or inhaled, orintestinal administration; parenteral delivery, including intradermal,transdermal (topical), intramuscular, subcutaneous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intravenous, intraperitoneal, and/or intranasal administration.

Alternately or additionally, liposomally encapsulated mRNAs andcompositions of the invention may be administered in a local rather thansystemic manner, for example, via injection of the pharmaceuticalcomposition directly into a targeted tissue, preferably in a sustainedrelease formulation. Local delivery can be affected in various ways,depending on the tissue to be targeted. For example, aerosols containingcompositions of the present invention can be inhaled (for nasal,tracheal, or bronchial delivery); compositions of the present inventioncan be injected into the site of injury, disease manifestation, or pain,for example; compositions can be provided in lozenges for oral,tracheal, or esophageal application; can be supplied in liquid, tabletor capsule form for administration to the stomach or intestines, can besupplied in suppository form for rectal or vaginal application; or caneven be delivered to the eye by use of creams, drops, or even injection.Formulations containing provided compositions complexed with therapeuticmolecules or ligands can even be surgically administered, for example inassociation with a polymer or other structure or substance that canallow the compositions to diffuse from the site of implantation tosurrounding cells. Alternatively, they can be applied surgically withoutthe use of polymers or supports.

In some embodiments, provided liposomes and/or compositions areformulated such that they are suitable for extended-release of the mRNAcontained therein. Such extended-release compositions may beconveniently administered to a subject at extended dosing intervals. Forexample, in one embodiment, the compositions of the present inventionare administered to a subject twice day, daily or every other day. In apreferred embodiment, the compositions of the present invention areadministered to a subject twice a week, once a week, every ten days,every two weeks, every three weeks, or more preferably every four weeks,once a month, every six weeks, every eight weeks, every other month,every three months, every four months, every six months, every eightmonths, every nine months or annually. Also contemplated arecompositions and liposomes which are formulated for depot administration(e.g., intramuscularly, subcutaneously, intravitreally) to eitherdeliver or release a mRNA over extended periods of time. Preferably, theextended-release means employed are combined with modifications made tothe mRNA to enhance stability.

Also contemplated herein are lyophilized pharmaceutical compositionscomprising one or more of the liposomes disclosed herein and relatedmethods for the use of such compositions as disclosed for example, inU.S. Provisional Application No. 61/494,882, filed Jun. 8, 2011, theteachings of which are incorporated herein by reference in theirentirety. For example, lyophilized pharmaceutical compositions accordingto the invention may be reconstituted prior to administration or can bereconstituted in vivo. For example, a lyophilized pharmaceuticalcomposition can be formulated in an appropriate dosage form (e.g., anintradermal dosage form such as a disk, rod or membrane) andadministered such that the dosage form is rehydrated over time in vivoby the individual's bodily fluids.

Provided liposomes and compositions may be administered to any desiredtissue. In some embodiments, the mRNA delivered by provided liposomes orcompositions is expressed in the tissue in which the liposomes and/orcompositions were administered. In some embodiments, the mRNA deliveredis expressed in a tissue different from the tissue in which theliposomes and/or compositions were administered Exemplary tissues inwhich delivered mRNA may be delivered and/or expressed include, but arenot limited to the liver, kidney, heart, spleen, serum, brain, skeletalmuscle, lymph nodes, skin, and/or cerebrospinal fluid.

According to various embodiments, the timing of expression of deliveredmRNAs can be tuned to suit a particular medical need. In someembodiments, the expression of the protein encoded by delivered mRNA isdetectable 1, 2, 3, 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, and/or 72hours in serum or target tissues after a single administration ofprovided liposomes or compositions. In some embodiments, the expressionof the protein encoded by the mRNA is detectable 1 day, 2 days, 3 days,4 days, 5 days, 6 days, and/or 7 days in serum or target tissues after asingle administration of provided liposomes or compositions. In someembodiments, the expression of the protein encoded by the mRNA isdetectable 1 week, 2 weeks, 3 weeks, and/or 4 weeks in serum or targettissues after a single administration of provided liposomes orcompositions. In some embodiments, the expression of the protein encodedby the mRNA is detectable after a month or longer after a singleadministration of provided liposomes or compositions.

The present invention can be used to deliver mRNA at various doses. Insome embodiments, an mRNA is administered at a dose ranging from about0.1-5.0 mg/kg body weight, for example about 0.1-4.5, 0.1-4.0, 0.1-3.5,0.1-3.0, 0.1-2.5, 0.1-2.0, 0.1-1.5, 0.1-1.0, 0.1-0.5, 0.1-0.3, 0.3-5.0,0.3-4.5, 0.3-4.0, 0.3-3.5, 0.3-3.0, 0.3-2.5, 0.3-2.0, 0.3-1.5, 0.3-1.0,0.3-0.5, 0.5-5.0, 0.5-4.5, 0.5-4.0, 0.5-3.5, 0.5-3.0, 0.5-2.5, 0.5-2.0,0.5-1.5, or 0.5-1.0 mg/kg body weight. In some embodiments, an mRNA isadministered at a dose of or less than about 5.0, 4.5, 4.0, 3.5, 3.0,2.5, 2.0, 1.5, 1.0, 0.8, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mg/kg bodyweight.

EXAMPLES

While certain compounds, compositions and methods of the presentinvention have been described with specificity in accordance withcertain embodiments, the following examples serve only to illustrate thecompounds of the invention and are not intended to limit the same.

Example 1. Exemplary Liposome Formulations for mRNA Delivery andExpression

This example provides exemplary liposome formulations incorporating thecationic lipids described in this application, for example, cKK-E12, foreffective delivery and expression of mRNA encoding therapeutic proteinsin vivo.

Lipid Materials

In general, the formulations described herein are based on amulti-component lipid mixture of varying ratios employing one or morecationic lipids, one or more helper lipids (e.g., non-cationic lipidsand/or cholesterol-based lipids), and one or more PEGylated lipidsdesigned to encapsulate various nucleic acid-based materials. As anon-limiting example, cKK-E12(3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione) isused in various formulations described herein. Exemplary helper lipidsinclude one or more of DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), cholesterol, etc.Exemplary PEGylated lipids include a poly(ethylene) glycol chain of upto 5 kDa in length covalently attached to a lipid with alkyl chain(s) ofC₆-C₂₀ length, for example, PEG-2K. As non-limiting examples, liposomeformulations used in various examples described herein include cKK-E12,DOPE, cholesterol and DMG-PEG2K at various ratios. For example, in somecases, the ratio of cKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately40:30:20:10 by weight. In other cases, the ratio ofcKK-E12:DOPE:cholesterol:DMG-PEG2K is approximately 40:32:25:3 byweight. Unless otherwise specified, the below Examples include a mixturein the ratio of cKK-E 12:DOPE:cholesterol:DMG-PEG2K of approximately40:30:25:5 by weight.

Messenger RNA Material

The formulations described herein may be used to deliver any mRNA, inparticular, therapeutic mRNA. As used herein, a therapeutic mRNA refersto an mRNA that encodes a therapeutic protein. The formulationsdescribed herein can also be used to deliver any modified or unmodifiedmRNA, or mRNA with naturally occurring sequences or codon-optimized.

As non-limiting examples, human Factor IX (FIX), codon-optimized FireflyLuciferase (FFL), codon-optimized human argininosuccinate synthetase(ASS1) messenger RNA, codon-optimized human Survival of Motor Neuron 1(SMN) mRNA were synthesized by in vitro transcription from a plasmid DNAtemplate encoding the gene, which was followed by the addition of a 5′cap structure (Cap 1) (Fechter, P.; Brownlee, G. G. “Recognition of mRNAcap structures by viral and cellular proteins” J. Gen. Virology 2005,86, 1239-1249) and a 3′ poly(A) tail of, e.g., approximately 250nucleotides in length (SEQ ID NO: 15) as determined by gelelectrophoresis. Typically, 5′ and 3′ untranslated regions (UTR) arepresent in each mRNA product and are represented as X and Y,respectively. Example 5′ and 3′ UTR sequences are described below. Theexemplary sequences of FIX, ASS1, and FFL mRNA used in the examplesherein are listed below. Also shown are the 5′ and 3′ UTR sequences.

Human Factor IX (FIX) mRNA:  (SEQ ID NO.: 1)XAUGCAGCGCGUGAACAUGAUCAUGGCAGAAUCACCAGGCCUCAUCACCAUCUGCCUUUUAGGAUAUCUACUCAGUGCUGAAUGUACAGUUUUUCUUGAUCAUGAAAACGCCAACAAAAUUCUGAGGCGGAGAAGGAGGUAUAAUUCAGGUAAAUUGGAAGAGUUUGUUCAAGGGAACCUUGAGAGAGAAUGUAUGGAAGAAAAGUGUAGUUUUGAAGAAGCACGAGAAGUUUUUGAAAACACUGAAAGAACAACUGAAUUUUGGAAGCAGUAUGUUGAUGGAGAUCAGUGUGAGUCCAAUCCAUGUUUAAAUGGCGGCAGUUGCAAGGAUGACAUUAAUUCCUAUGAAUGUUGGUGUCCCUUUGGAUUUGAAGGAAAGAACUGUGAAUUAGAUGUAACAUGUAACAUUAAGAAUGGCAGAUGCGAGCAGUUUUGUAAAAAUAGUGCUGAUAACAAGGUGGUUUGCUCCUGUACUGAGGGAUAUCGACUUGCAGAAAACCAGAAGUCCUGUGAACCAGCAGUGCCAUUUCCAUGUGGAAGAGUUUCUGUUUCACAAACUUCUAAGCUCACCCGUGCUGAGGCUGUUUUUCCUGAUGUGGACUAUGUAAAUUCUACUGAAGCUGAAACCAUUUUGGAUAACAUCACUCAAAGCACCCAAUCAUUUAAUGACUUCACUCGGGUUGUUGGUGGAGAAGAUGCCAAACCAGGUCAAUUCCCUUGGCAGGUUGUUUUGAAUGGUAAAGUUGAUGCAUUCUGUGGAGGCUCUAUCGUUAAUGAAAAAUGGAUUGUAACUGCUGCCCACUGUGUUGAAACUGGUGUUAAAAUUACAGUUGUCGCAGGUGAACAUAAUAUUGAGGAGACAGAACAUACAGAGCAAAAGCGAAAUGUGAUUCGAAUUAUUCCUCACCACAACUACAAUGCAGCUAUUAAUAAGUACAACCAUGACAUUGCCCUUCUGGAACUGGACGAACCCUUAGUGCUAAACAGCUACGUUACACCUAUUUGCAUUGCUGACAAGGAAUACACGAACAUCUUCCUCAAAUUUGGAUCUGGCUAUGUAAGUGGCUGGGGAAGAGUCUUCCACAAAGGGAGAUCAGCUUUAGUUCUUCAGUACCUUAGAGUUCCACUUGUUGACCGAGCCACAUGUCUUCGAUCUACAAAGUUCACCAUCUAUAACAACAUGUUCUGUGCUGGCUUCCAUGAAGGAGGUAGAGAUUCAUGUCAAGGAGAUAGUGGGGGACCCCAUGUUACUGAAGUGGAAGGGACCAGUUUCUUAACUGGAAUUAUUAGCUGGGGUGAAGAGUGUGCAAUGAAAGGCAAAUAUGGAAUAUAUACCAAGGUAUCCCGGUAUGUCAACUGGAUUAAGGAAAAAACAAAGCUCACUUAAY Codon-Optimized Human Argininosuccinate Synthetase (ASS1) mRNA: (SEQ ID NO.: 2) XAUGAGCAGCAAGGGCAGCGUGGUGCUGGCCUACAGCGGCGGCCUGGACACCAGCUGCAUCCUGGUGUGGCUGAAGGAGCAGGGCUACGACGUGAUCGCCUACCUGGCCAACAUCGGCCAGAAGGAGGACUUCGAGGAGGCCCGCAAGAAGGCCCUGAAGCUGGGCGCCAAGAAGGUGUUCAUCGAGGACGUGAGCCGCGAGUUCGUGGAGGAGUUCAUCUGGCCCGCCAUCCAGAGCAGCGCCCUGUACGAGGACCGCUACCUGCUGGGCACCAGCCUGGCCCGCCCCUGCAUCGCCCGCAAGCAGGUGGAGAUCGCCCAGCGCGAGGGCGCCAAGUACGUGAGCCACGGCGCCACCGGCAAGGGCAACGACCAGGUGCGCUUCGAGCUGAGCUGCUACAGCCUGGCCCCCCAGAUCAAGGUGAUCGCCCCCUGGCGCAUGCCCGAGUUCUACAACCGCUUCAAGGGCCGCAACGACCUGAUGGAGUACGCCAAGCAGCACGGCAUCCCCAUCCCCGUGACCCCCAAGAACCCCUGGAGCAUGGACGAGAACCUGAUGCACAUCAGCUACGAGGCCGGCAUCCUGGAGAACCCCAAGAACCAGGCCCCCCCCGGCCUGUACACCAAGACCCAGGACCCCGCCAAGGCCCCCAACACCCCCGACAUCCUGGAGAUCGAGUUCAAGAAGGGCGUGCCCGUGAAGGUGACCAACGUGAAGGACGGCACCACCCACCAGACCAGCCUGGAGCUGUUCAUGUACCUGAACGAGGUGGCCGGCAAGCACGGCGUGGGCCGCAUCGACAUCGUGGAGAACCGCUUCAUCGGCAUGAAGAGCCGCGGCAUCUACGAGACCCCCGCCGGCACCAUCCUGUACCACGCCCACCUGGACAUCGAGGCCUUCACCAUGGACCGCGAGGUGCGCAAGAUCAAGCAGGGCCUGGGCCUGAAGUUCGCCGAGCUGGUGUACACCGGCUUCUGGCACAGCCCCGAGUGCGAGUUCGUGCGCCACUGCAUCGCCAAGAGCCAGGAGCGCGUGGAGGGCAAGGUGCAGGUGAGCGUGCUGAAGGGCCAGGUGUACAUCCUGGGCCGCGAGAGCCCCCUGAGCCUGUACAACGAGGAGCUGGUGAGCAUGAACGUGCAGGGCGACUACGAGCCCACCGACGCCACCGGCUUCAUCAACAUCAACAGCCUGCGCCUGAAGGAGUACCACCGCCUGCAGAGCAAGGUGACCGCCAAGUGAYCodon-Optimized Firefly Luciferase (FFL) mRNA:  (SEQ ID NO.: 3)XAUGGAAGAUGCCAAAAACAUUAAGAAGGGCCCAGCGCCAUUCUACCCACUCGAAGACGGGACCGCCGGCGAGCAGCUGCACAAAGCCAUGAAGCGCUACGCCCUGGUGCCCGGCACCAUCGCCUUUACCGACGCACAUAUCGAGGUGGACAUUACCUACGCCGAGUACUUCGAGAUGAGCGUUCGGCUGGCAGAAGCUAUGAAGCGCUAUGGGCUGAAUACAAACCAUCGGAUCGUGGUGUGCAGCGAGAAUAGCUUGCAGUUCUUCAUGCCCGUGUUGGGUGCCCUGUUCAUCGGUGUGGCUGUGGCCCCAGCUAACGACAUCUACAACGAGCGCGAGCUGCUGAACAGCAUGGGCAUCAGCCAGCCCACCGUCGUAUUCGUGAGCAAGAAAGGGCUGCAAAAGAUCCUCAACGUGCAAAAGAAGCUACCGAUCAUACAAAAGAUCAUCAUCAUGGAUAGCAAGACCGACUACCAGGGCUUCCAAAGCAUGUACACCUUCGUGACUUCCCAUUUGCCACCCGGCUUCAACGAGUACGACUUCGUGCCCGAGAGCUUCGACCGGGACAAAACCAUCGCCCUGAUCAUGAACAGUAGUGGCAGUACCGGAUUGCCCAAGGGCGUAGCCCUACCGCACCGCACCGCUUGUGUCCGAUUCAGUCAUGCCCGCGACCCCAUCUUCGGCAACCAGAUCAUCCCCGACACCGCUAUCCUCAGCGUGGUGCCAUUUCACCACGGCUUCGGCAUGUUCACCACGCUGGGCUACUUGAUCUGCGGCUUUCGGGUCGUGCUCAUGUACCGCUUCGAGGAGGAGCUAUUCUUGCGCAGCUUGCAAGACUAUAAGAUUCAAUCUGCCCUGCUGGUGCCCACACUAUUUAGCUUCUUCGCUAAGAGCACUCUCAUCGACAAGUACGACCUAAGCAACUUGCACGAGAUCGCCAGCGGCGGGGCGCCGCUCAGCAAGGAGGUAGGUGAGGCCGUGGCCAAACGCUUCCACCUACCAGGCAUCCGCCAGGGCUACGGCCUGACAGAAACAACCAGCGCCAUUCUGAUCACCCCCGAAGGGGACGACAAGCCUGGCGCAGUAGGCAAGGUGGUGCCCUUCUUCGAGGCUAAGGUGGUGGACUUGGACACCGGUAAGACACUGGGUGUGAACCAGCGCGGCGAGCUGUGCGUCCGUGGCCCCAUGAUCAUGAGCGGCUACGUUAACAACCCCGAGGCUACAAACGCUCUCAUCGACAAGGACGGCUGGCUGCACAGCGGCGACAUCGCCUACUGGGACGAGGACGAGCACUUCUUCAUCGUGGACCGGCUGAAGAGCCUGAUCAAAUACAAGGGCUACCAGGUAGCCCCAGCCGAACUGGAGAGCAUCCUGCUGCAACACCCCAACAUCUUCGACGCCGGGGUCGCCGGCCUGCCCGACGACGAUGCCGGCGAGCUGCCCGCCGCAGUCGUCGUGCUGGAACACGGUAAAACCAUGACCGAGAAGGAGAUCGUGGACUAUGUGGCCAGCCAGGUUACAACCGCCAAGAAGCUGCGCGGUGGUGUUGUGUUCGUGGACGAGGUGCCUAAAGGACUGACCGGCAAGUUGGACGCCCGCAAGAUCCGCGAGAUUCUCAUUAAGGCCAAGAAGGGCGGCAAGAUCGCCGU GUAAY Codon-Optimized Human Survival of Motor Neuron 1(SMN) mRNA: (SEQ ID NO: 4) XAUGGCCAUGAGCAGCGGAGGCAGCGGCGGAGGAGUGCCCGAGCAGGAGGACAGCGUGCUGUUCAGGAGAGGCACCGGCCAGAGCGAUGACAGCGAUAUCUGGGACGAUACCGCUCUGAUCAAGGCCUACGACAAGGCCGUGGCCAGCUUCAAGCACGCCCUGAAAAACGGCGACAUCUGCGAGACCAGCGGCAAGCCCAAGACAACCCCCAAGAGAAAGCCCGCCAAGAAGAAUAAGAGCCAGAAAAAGAACACCGCCGCCAGCCUGCAGCAGUGGAAGGUGGGCGACAAGUGCAGCGCCAUCUGGAGCGAGGACGGCUGCAUCUACCCCGCCACCAUCGCCAGCAUCGACUUCAAGAGAGAGACCUGCGUGGUCGUGUACACCGGCUACGGCAACAGAGAGGAGCAGAACCUGAGCGACCUGCUGAGCCCCAUUUGUGAGGUGGCCAAUAACAUCGAACAGAACGCCCAGGAGAACGAGAAUGAAAGCCAGGUGAGCACCGACGAGAGCGAGAACAGCAGAUCUCCUGGCAACAAGAGCGACAACAUCAAGCCUAAGUCUGCCCCUUGGAACAGCUUCCUGCCCCCUCCUCCACCCAUGCCCGGACCCAGACUGGGACCCGGAAAACCUGGCCUGAAGUUCAACGGACCACCUCCCCCUCCACCUCCUCCCCCACCUCAUCUCCUGAGCUGCUGGCUGCCACCCUUCCCCAGCGGACCCCCUAUCAUCCCACCACCCCCUCCCAUCUGCCCCGACAGCCUGGACGACGCCGAUGCCCUGGGCAGCAUGCUGAUCAGCUGGUACAUGAGCGGCUACCACACAGGAUACUACAUGGGCUUCAGACAGAACCAGAAGGAGGGCAGAUGCUCCCACUCCCUGAACUGAY 5′ and 3′ UTR Sequences  X (5′UTR Sequence) = (SEQ ID NO.: 5)GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG  Y (3′ UTR Sequence) =(SEQ ID NO.: 6) CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUC AAGCU  or (SEQ ID NO.: 7) GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCA AAGCU C-terminal His 10 Codon-Optimized Human CFTR mRNA (″His₁₀″disclosed as SEQ ID NO: 11):  (SEQ ID NO.: 8)XAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUCACCAUCACCAUCACCAUCACCAUCACCAUUAAY  Codon-Optimized Human CF TR mRNA: (SEQ ID NO.: 9) XAUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAAY Codon Optimized Human CFTR mRNA coding sequencewith a Growth Hormone Leader Sequence (italisized and underlined) (SEQ ID NO: 10) AUGGCCACUGGAUCAAGAACCUCACUGCUGCUCGCUUUUGGACUGCUUUGCCUGCCCUGGUUGCAAGAAGGAUCGGCUUUCCCGACCAUCCCACUCUCC AUGCAGCGGUCCCCGCUCGAAAAGGCCAGUGUCGUGUCCAAACUCUUCUUCUCAUGGACUCGGCCUAUCCUUAGAAAGGGGUAUCGGCAGAGGCUUGAGUUGUCUGACAUCUACCAGAUCCCCUCGGUAGAUUCGGCGGAUAACCUCUCGGAGAAGCUCGAACGGGAAUGGGACCGCGAACUCGCGUCUAAGAAAAACCCGAAGCUCAUCAACGCACUGAGAAGGUGCUUCUUCUGGCGGUUCAUGUUCUACGGUAUCUUCUUGUAUCUCGGGGAGGUCACAAAAGCAGUCCAACCCCUGUUGUUGGGUCGCAUUAUCGCCUCGUACGACCCCGAUAACAAAGAAGAACGGAGCAUCGCGAUCUACCUCGGGAUCGGACUGUGUUUGCUUUUCAUCGUCAGAACACUUUUGUUGCAUCCAGCAAUCUUCGGCCUCCAUCACAUCGGUAUGCAGAUGCGAAUCGCUAUGUUUAGCUUGAUCUACAAAAAGACACUGAAACUCUCGUCGCGGGUGUUGGAUAAGAUUUCCAUCGGUCAGUUGGUGUCCCUGCUUAGUAAUAACCUCAACAAAUUCGAUGAGGGACUGGCGCUGGCACAUUUCGUGUGGAUUGCCCCGUUGCAAGUCGCCCUUUUGAUGGGCCUUAUUUGGGAGCUGUUGCAGGCAUCUGCCUUUUGUGGCCUGGGAUUUCUGAUUGUGUUGGCAUUGUUUCAGGCUGGGCUUGGGCGGAUGAUGAUGAAGUAUCGCGACCAGAGAGCGGGUAAAAUCUCGGAAAGACUCGUCAUCACUUCGGAAAUGAUCGAAAACAUCCAGUCGGUCAAAGCCUAUUGCUGGGAAGAAGCUAUGGAGAAGAUGAUUGAAAACCUCCGCCAAACUGAGCUGAAACUGACCCGCAAGGCGGCGUAUGUCCGGUAUUUCAAUUCGUCAGCGUUCUUCUUUUCCGGGUUCUUCGUUGUCUUUCUCUCGGUUUUGCCUUAUGCCUUGAUUAAGGGGAUUAUCCUCCGCAAGAUUUUCACCACGAUUUCGUUCUGCAUUGUAUUGCGCAUGGCAGUGACACGGCAAUUUCCGUGGGCCGUGCAGACAUGGUAUGACUCGCUUGGAGCGAUCAACAAAAUCCAAGACUUCUUGCAAAAGCAAGAGUACAAGACCCUGGAGUACAAUCUUACUACUACGGAGGUAGUAAUGGAGAAUGUGACGGCUUUUUGGGAAGAGGGUUUUGGAGAACUGUUUGAGAAAGCAAAGCAGAAUAACAACAACCGCAAGACCUCAAAUGGGGACGAUUCCCUGUUUUUCUCGAACUUCUCCCUGCUCGGAACACCCGUGUUGAAGGACAUCAAUUUCAAGAUUGAGAGGGGACAGCUUCUCGCGGUAGCGGGAAGCACUGGUGCGGGAAAAACUAGCCUCUUGAUGGUGAUUAUGGGGGAGCUUGAGCCCAGCGAGGGGAAGAUUAAACACUCCGGGCGUAUCUCAUUCUGUAGCCAGUUUUCAUGGAUCAUGCCCGGAACCAUUAAAGAGAACAUCAUUUUCGGAGUAUCCUAUGAUGAGUACCGAUACAGAUCGGUCAUUAAGGCGUGCCAGUUGGAAGAGGACAUUUCUAAGUUCGCCGAGAAGGAUAACAUCGUCUUGGGAGAAGGGGGUAUUACAUUGUCGGGAGGGCAGCGAGCGCGGAUCAGCCUCGCGAGAGCGGUAUACAAAGAUGCAGAUUUGUAUCUGCUUGAUUCACCGUUUGGAUACCUCGACGUAUUGACAGAAAAAGAAAUCUUCGAGUCGUGCGUGUGUAAACUUAUGGCUAAUAAGACGAGAAUCCUGGUGACAUCAAAAAUGGAACACCUUAAGAAGGCGGACAAGAUCCUGAUCCUCCACGAAGGAUCGUCCUACUUUUACGGCACUUUCUCAGAGUUGCAAAACUUGCAGCCGGACUUCUCAAGCAAACUCAUGGGGUGUGACUCAUUCGACCAGUUCAGCGCGGAACGGCGGAACUCGAUCUUGACGGAAACGCUGCACCGAUUCUCGCUUGAGGGUGAUGCCCCGGUAUCGUGGACCGAGACAAAGAAGCAGUCGUUUAAGCAGACAGGAGAAUUUGGUGAGAAAAGAAAGAACAGUAUCUUGAAUCCUAUUAACUCAAUUCGCAAGUUCUCAAUCGUCCAGAAAACUCCACUGCAGAUGAAUGGAAUUGAAGAGGAUUCGGACGAACCCCUGGAGCGCAGGCUUAGCCUCGUGCCGGAUUCAGAGCAAGGGGAGGCCAUUCUUCCCCGGAUUUCGGUGAUUUCAACCGGACCUACACUUCAGGCGAGGCGAAGGCAAUCCGUGCUCAACCUCAUGACGCAUUCGGUAAACCAGGGGCAAAACAUUCACCGCAAAACGACGGCCUCAACGAGAAAAGUGUCACUUGCACCCCAGGCGAAUUUGACUGAACUCGACAUCUACAGCCGUAGGCUUUCGCAAGAAACCGGACUUGAGAUCAGCGAAGAAAUCAAUGAAGAAGAUUUGAAAGAGUGUUUCUUUGAUGACAUGGAAUCAAUCCCAGCGGUGACAACGUGGAACACAUACUUGCGUUACAUCACGGUGCACAAGUCCUUGAUUUUCGUCCUCAUCUGGUGUCUCGUGAUCUUUCUCGCUGAGGUCGCAGCGUCACUUGUGGUCCUCUGGCUGCUUGGUAAUACGCCCUUGCAAGACAAAGGCAAUUCUACACACUCAAGAAACAAUUCCUAUGCCGUGAUUAUCACUUCUACAAGCUCGUAUUACGUGUUUUACAUCUACGUAGGAGUGGCCGACACUCUGCUCGCGAUGGGUUUCUUCCGAGGACUCCCACUCGUUCACACGCUUAUCACUGUCUCCAAGAUUCUCCACCAUAAGAUGCUUCAUAGCGUACUGCAGGCUCCCAUGUCCACCUUGAAUACGCUCAAGGCGGGAGGUAUUUUGAAUCGCUUCUCAAAAGAUAUUGCAAUUUUGGAUGACCUUCUGCCCCUGACGAUCUUCGACUUCAUCCAGUUGUUGCUGAUCGUGAUUGGGGCUAUUGCAGUAGUCGCUGUCCUCCAGCCUUACAUUUUUGUCGCGACCGUUCCGGUGAUCGUGGCGUUUAUCAUGCUGCGGGCCUAUUUCUUGCAGACGUCACAGCAGCUUAAGCAACUGGAGUCUGAAGGGAGGUCGCCUAUCUUUACGCAUCUUGUGACCAGUUUGAAGGGAUUGUGGACGUUGCGCGCCUUUGGCAGGCAGCCCUACUUUGAAACACUGUUCCACAAAGCGCUGAAUCUCCAUACGGCAAAUUGGUUUUUGUAUUUGAGUACCCUCCGAUGGUUUCAGAUGCGCAUUGAGAUGAUUUUUGUGAUCUUCUUUAUCGCGGUGACUUUUAUCUCCAUCUUGACCACGGGAGAGGGCGAGGGACGGGUCGGUAUUAUCCUGACACUCGCCAUGAACAUUAUGAGCACUUUGCAGUGGGCAGUGAACAGCUCGAUUGAUGUGGAUAGCCUGAUGAGGUCCGUUUCGAGGGUCUUUAAGUUCAUCGACAUGCCGACGGAGGGAAAGCCCACAAAAAGUACGAAACCCUAUAAGAAUGGGCAAUUGAGUAAGGUAAUGAUCAUCGAGAACAGUCACGUGAAGAAGGAUGACAUCUGGCCUAGCGGGGGUCAGAUGACCGUGAAGGACCUGACGGCAAAAUACACCGAGGGAGGGAACGCAAUCCUUGAAAACAUCUCGUUCAGCAUUAGCCCCGGUCAGCGUGUGGGGUUGCUCGGGAGGACCGGGUCAGGAAAAUCGACGUUGCUGUCGGCCUUCUUGAGACUUCUGAAUACAGAGGGUGAGAUCCAGAUCGACGGCGUUUCGUGGGAUAGCAUCACCUUGCAGCAGUGGCGGAAAGCGUUUGGAGUAAUCCCCCAAAAGGUCUUUAUCUUUAGCGGAACCUUCCGAAAGAAUCUCGAUCCUUAUGAACAGUGGUCAGAUCAAGAGAUUUGGAAAGUCGCGGACGAGGUUGGCCUUCGGAGUGUAAUCGAGCAGUUUCCGGGAAAACUCGACUUUGUCCUUGUAGAUGGGGGAUGCGUCCUGUCGCAUGGGCACAAGCAGCUCAUGUGCCUGGCGCGAUCCGUCCUCUCUAAAGCGAAAAUUCUUCUCUUGGAUGAACCUUCGGCCCAUCUGGACCCGGUAACGUAUCAGAUCAUCAGAAGGACACUUAAGCAGGCGUUUGCCGACUGCACGGUGAUUCUCUGUGAGCAUCGUAUCGAGGCCAUGCUCGAAUGCCAGCAAUUUCUUGUCAUCGAAGAGAAUAAGGUCCGCCAGUACGACUCCAUCCAGAAGCUGCUUAAUGAGAGAUCAUUGUUCCGGCAGGCGAUUUCACCAUCCGAUAGGGUGAAACUUUUUCCACACAGAAAUUCGUCGAAGUGCAAGUCCAAACCGCAGAUCGCGGCCUUGAAAGAAGAGACUGAAGAAGAAGUUCAAGACACGCGUCUUUAA

Aliquots of 50 mg/mL ethanolic solutions of cKK-E12, DOPE, Chol andDMG-PEG2K were mixed in a molar ratio of 40:30:25:5 and diluted withethanol to 3 mL final volume. Separately, an aqueous buffered solution(10 mM citrate/150 mM NaCl, pH 4.5) of FIX, ASS1, or FFL mRNA wasprepared from a 1 mg/mL stock. The lipid solution was injected rapidlyinto the aqueous mRNA solution and shaken to yield a final suspension in20% ethanol. The resulting nanoparticle suspension was filtered,diafiltrated with 1×PBS (pH 7.4), concentrated and stored at 2-8° C. Thefinal concentration of FIX mRNA was approximately 0.77 mg/mL FIX mRNA(encapsulated), Z_(ave)=76 nm, PDI=0.08. The final concentration of ASS1mRNA was approximately 0.64 mg/mL ASS1 mRNA (encapsulated), Z_(ave)=78nm (Dv(50)=46 nm; Dv(90)=96 nm). The final concentration of FFL mRNA wasapproximately 1.31 mg/mL FFL mRNA (encapsulated), Z_(ave)=75 nm,PDI-0.11. The final concentration of SMN mRNA was approximately 1.85mg/mL SMN mRNA (encapsulated). Average particle size (Z_(ave))=71 nm,(particle size for 50% of particles was 44 nm or less (Dv(50))=44 nm;and the particle size for 90% of the particles was 93n or less(Dv(90)=93 nm)).

Example 2. Administration of mRNA-Loaded Liposome Nanoparticles

This example illustrates exemplary methods of administering mRNA-loadedliposome nanoparticles and methods for analyzing delivered mRNA andsubsequently expressed protein in various target tissues in vivo.

All studies were performed using male CD-1 mice of approximately 6-8weeks of age at the beginning of each experiment. Samples wereintroduced by a single bolus tail-vein injection of an equivalent totaldose of 1.0 mg/kg (or otherwise specified) of encapsulated FIX, FFL orASS1 mRNA. Mice were sacrificed and perfused with saline at thedesignated time points.

Various organ tissues such as the liver, spleen, kidney and heart ofeach mouse was harvested, apportioned into separate parts, and stored ineither 10% neutral buffered formalin or snap-frozen and stored at −80°C. for analysis.

All animals were euthanized by CO₂ asphyxiation at designated timepoints post dose administration (±5%) followed by thoracotomy andterminal cardiac blood collection. Whole blood (maximal obtainablevolume) was collected via cardiac puncture on euthanized animals intoserum separator tubes, allowed to clot at room temperature for at least30 minutes, centrifuged at 22° C.±5° C. at 9300 g for 10 minutes, andthe serum extracted. For interim blood collections, approximately 40-50μL of whole blood was collected via facial vein puncture or tail snip.Samples collected from non-treatment animals were used as a baselineASS1 levels for comparison to study animals.

Enzyme-Linked Immunosorbant Assay (ELISA) Analysis

A. Human FIX ELISA

Quantification of FIX protein was performed following proceduresreported for human FIX ELISA kit (AssayMax, Assay Pro, Catalog#EF1009-1).

B. Human ASS1 ELISA

Standard ELISA procedures were followed employing mouse anti-ASS12D1-2E12 IgG as the capture antibody with rabbit anti-ASS1 #3285 IgG asthe secondary (detection) antibody (Shire Human Genetic Therapies).Horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG was usedfor activation of the 3,3′,5,5′-tetramethylbenzidine (TMB) substratesolution. The reaction was quenched using 2N H2SO4 after 20 minutes.Detection was monitored via absorption (450 nm) on a Molecular DeviceSpectraMax instrument. Untreated mouse serum and organs and human ASS1protein were used as negative and positive controls, respectively.

IVIS Bioluminometer Measurements

To visual luminescence in treated mice, several steps were followed.Anesthesia using isoflurane vaporizer at 1-3% (usually @2.5%) wasinitially employed. Using a microsprayer, 50 μL/animal of luciferin inPBS was administered at 60 mg/mL via intratracheal/intranasal. Luciferinwas allowed to distribute for 5-10 minutes. Animals were placed in anisoflurane chamber until anesthetized. Anesthetized animals were placedinto the IVIS imaging chamber at dorsal recumbency and positioned intothe manifold. Pictures of mice were taken. In these Examples, theacquisition settings providing highest sensitivity were: camera heightat D level, F/Stop at f1, binning at high resolution, and exposure timeat 5 minutes. Exposures were repeated up to 3 times (5, 10 and 15minutes post Luciferin Injection).

In Situ Hybridization (ISH) Analysis

In situ hybridization was performed using “ZZ” probe technology. Probeswere generated based on codon-optimized sequence of human messenger RNA.Tissues were fixed for 24-48 hours in 10% neutral buffered formalin andembedded in paraffin. Positive detection of desired mRNA was achievedthrough 6 consecutive amplification steps followed by chromagenicvisualization using 3,3′-diaminobenzidine (DAB). Positive signal wascompared to that of untreated mouse.

Example 3. Highly Effective In Vivo Production of Therapeutic Proteins

This example demonstrates highly efficient and sustained production ofproteins encoded by mRNA delivered by liposomes incorporating thecationic lipids described herein (e.g., cKK-E12) in serum and variousorgan tissues.

In Vivo Human FIX Protein Production Results

The production of human FIX protein via hFIX mRNA-loaded cKK-E12-basedlipid nanoparticles was tested in CD-1 mice as a single, bolusintravenous injection. FIG. 1 represents the amount of human FIX proteindetected via ELISA when treating mice with human FIX mRNA-loadedcKK-E12-based lipid nanoparticles as compared to a C12-200-based lipidnanoparticle encapsulating hFIX mRNA. The mice were sacrificedtwenty-four hours post-injection and organs were harvested (as describedabove).

C12-200-based lipid nanoparticles have been shown to be an effectivevehicle to deliver and express mRNA in vivo (see, PCT ApplicationPublication NO. WO2012170930, the disclosure of which is herebyincorporated by reference). Surprisingly, as represented in FIG. 1,cKK-E12 based lipid nanoparticles are even more effective in deliveringhuman FIX mRNA in vivo, resulting in close to 50% higher proteinexpression detected in the plasma of the treated mice, as compared toC12-200-based lipid nanoparticles.

FIG. 2 shows the results of a dose-response experiment as represented bythe amount of human FIX protein detected via ELISA when treating micewith human FIX mRNA-loaded cKK-E12-based lipid nanoparticles at variousdoses. The mice were bled at 6 hours and sacrificed twenty-four hourspost-injection and organs were harvested (as described above).

A clear dose response was achieved when measuring liver levels of humanFIX protein. The dosing range was from 0.10-3.0 mg/kg of encapsulatedhuman FIX mRNA. These data demonstrate the ability of the lipidnanoparticles to efficiently deliver messenger RNA, release the payloadand process this exogenous mRNA via translation to produce human FIXprotein, which is then subsequently secreted into the bloodstream.Levels of human FIX protein are well above therapeutic levels (>100ng/mL plasma) and surpass normal physiological levels (˜5 ug/mL plasma)when dosing at 1.0 mg/kg or greater. Further, the plasma residence timeof this human protein is sustained through at least 24 hours postadministration.

In Vivo Human ASS1 Protein Production Results

The production of human ASS1 protein via codon-optimized hASS1mRNA-loaded cKK-E12-based lipid nanoparticles was tested in CD-1 mice asa single, bolus intravenous injection. FIG. 3 represents the amount ofhuman ASS1 protein detected via ELISA when treating mice with human ASS1mRNA-loaded cKK-E12-based lipid nanoparticles at various doses. The micewere sacrificed twenty-four hours post-injection and organs wereharvested (as described above).

A clear dose response was achieved when measuring liver levels of humanASS1 protein. As shown in Table 5, the dosing range was from 0.10-2.0mg/kg of encapsulated human ASS1 mRNA in cKK-E12 lipid nanoparticles.These data demonstrate the ability of the lipid nanoparticles toaccumulate in the liver and release the mRNA payload and the liver toprocess this exogenous mRNA via translation to produce human ASS1protein.

TABLE 5 Raw values of human ASS1 protein as measured via ELISA analysis(as depicted in FIG. 1). Codon-optimized human ASS1 mRNA was deliveredvia cKK-E12-based lipid nanoparticles. Doses are based on encapsulatedASS1 mRNA. Values are depicted as nanogram of human ASS1 protein permilligram total protein in liver. Dose Encapsulated Human ASS1 mRNA(mg/kg) ASS1 Protein (ng/mg total protein) 0.10 BLD 0.30 BLD 0.60 5461.0 1388 2.0 3371 BLD = Below Limit of Detection for ELISA.

While the sensitivity of the ELISA has limitations at lower values,western blot analysis allows for clear visualization of the human ASS1protein at lower doses (0.3-3.0 mg/kg) (see FIG. 4). FIG. 4 depicts acomparison of human ASS1 protein levels in liver as a function of dosevia western blot analysis upon a single intravenous dose of human ASS1mRNA-encapsulated cKK-E12 lipid nanoparticles. CD1 mice were sacrificedat 24 hours post-administration and livers were harvested and analyzedas described above.

To further understand the ability of ASS1 mRNA-encapsulated lipidnanoparticles to facilitate the delivery of mRNA to selected organs(liver), a pharmacokinetic analysis was performed, monitoring human ASS1protein levels in the liver over a one week time period. FIG. 5 depictsthe quantity of human ASS1 protein detected in the liver at various timepoints up to 7 days after administration of human ASS1-loaded lipidnanoparticles (cKK-E12). This was accomplished as a single dose (1.0mg/kg encapsulated mRNA) given intravenously.

In this case, we observed a maximum serum level of human ASS1 protein atapproximately 24-48 hours post-administration. Measurable levels ofprotein were still observed 1 week post-administration as determined byboth ELISA and western blot (FIGS. 5 and 6, respectively). FIG. 6depicts a comparison of human ASS1 protein levels in liver over time viawestern blot analysis upon a single intravenous dose of human ASS1mRNA-encapsulated lipid nanoparticles (1.0 mg/kg dose).

Direct detection of the active pharmaceutical ingredient (ASS1 mRNA) inthe livers of the treated mice was achieved using in situ hybridization(ISH) based methods. As demonstrated in FIGS. 7 & 8, the exogenous humanASS1 messenger RNA could be detected in high levels at the earliest timepoint tested (30 minutes) and the signal remained strong for 48 hoursafter dosing. Further, human ASS1 mRNA was still detectable 72 hourspost-administration.

In addition to ISH, detection of the resulting human ASS1 protein wasachieved using immunohistochemical (IHC) means. Using a mouse monoclonalantibody (02D2-2E12) for specific binding, the presence of target humanASS1 protein in the cytoplasm of hepatocytes of treated livers can bereadily observed. FIG. 9 shows the immunohistochemical staining of humanASS1 protein in treated mouse livers 24 hours after administration.

In Vivo Delivery of FFL mRNA Via Nebulization

To assess whether additional routes of delivery were feasible, FFL mRNAwas encapsulated in cKK-E12 liposomes and those liposomes werenebulized. As shown in FIG. 10, it is possible to efficiently nebulizecKK-E12 based lipid nanoparticles encapsulating mRNA. FIG. 10 representsmice treated with luciferin 24 hours after exposure to nebulized FFLmRNA loaded cKK-E12 lipid nanoparticles.

Example 4. CNS Delivery of hSMN-1 mRNA

This example provides an exemplary cKK-E12 liposome formulations foreffective delivery and expression of mRNA in the CNS. Specifically, theexample demonstrates that delivery of human survival of motor neuron-1(hSMN-1) mRNA into various tissues of the brain and spinal cord.

Messenger RNA Material

Codon-optimized human Survival of Motor Neuron-1 (hSMN-1) messenger RNA(see SEQ ID NO: 4) was synthesized by in vitro transcription from aplasmid DNA template encoding the gene, which was followed by theaddition of a 5′ cap structure (Cap 1) (Fechter, P.; Brownlee, G. G.“Recognition of mRNA cap structures by viral and cellular proteins” J.Gen. Virology 2005, 86, 1239-1249) and a 3′ poly(A) tail ofapproximately 250 nucleotides in length (SEQ ID NO: 15) as determined bygel electrophoresis. The 5′ and 3′ untranslated regions present in eachmRNA product are represented as X and Y, respectively and defined asstated in Example 1.

Formulation Protocol

Lipid nanoparticles (LNP) were formed via standard ethanol injectionmethods (Ponsa, M.; Foradada, M.; Estelrich, J. “Liposomes obtained bythe ethanol injection method” Int. J. Pharm. 1993, 95, 51-56). For thevarious lipid components, a 50 mg/ml ethanolic stock solutions wasprepared and stored at −20° C. In preparation of the cKK-E12 lipidnanoparticle formulation listed in Table 6, each indicated lipidcomponent was added to an ethanol solution to achieve a predeterminedfinal concentration and molar ratio, and scaled to a 3 ml final volumeof ethanol. Separately, an aqueous buffered solution (10 mM citrate/150mM NaCl, pH 4.5) of hSMN-1 mRNA was prepared from a 1 mg/ml stock. Thelipid solution was injected rapidly into the aqueous mRNA solution andshaken to yield a final suspension in 20% ethanol. The resultingnanoparticle suspension was filtered and dialysed against 1×PBS (pH7.4), concentrated and stored between 2-8° C. SMN-1 mRNA concentrationwas determined via the Ribogreen assay (Invitrogen). Encapsulation ofmRNA was calculated by performing the Ribogreen assay with and withoutthe presence of 0.1% Triton-X 100. Particle sizes (dynamic lightscattering (DLS)) and zeta potentials were determined using a MalvernZetasizer instrument in 1×PBS and 1 mM KCl solutions, respectively.

TABLE 6 Exemplary cKK-E12 Lipid Nanoparticle formulation Final MolarmRNA Form- Ratio Concen- Zeta ulations Components of lipids trationParameters 1 cKK-E12 40:30:25:5 1.8 mg/ml Z_(ave) = 72 nm; DOPE Dv₍₅₀₎ =49 nm; Cholesterol Dv₍₉₀₎ = 90 nm DMG-PEG-2K hSMN-1 mRNA

Intrathecal Administration of mRNA Loaded Liposome Nanoparticles

All in vivo studies were performed using either rats or mice ofapproximately 6-8 weeks of age at the beginning of each experiment. Atthe start of the experiment, each animal was anesthetized withisoflurane (1-3%, to effect) by inhalation. Once anesthetized, eachanimal was shaved at the exact injection site (L4-L5 or L5-L6).Following insertion of the needle, reflexive flick of the tail was usedto indicate puncture of the dura and confirm intrathecal placement. Eachanimal received a single bolus intrathecal injection of the testformulation listed in Table 6. All animals were sacrificed 24 hours postinjection and perfused with saline.

Isolation of Organ Tissues for Analysis

All animals had the whole brain and spinal cord harvested. The brain wascut longitudinally and placed in one histology cassette per animal. Thewhole spinal cord was stored ambient in a 15 ml tube containing 10%neutral buffered formalin (NBF) for at least 24 hours and no more than72 hours before transfer into 70% histology grade alcohol solution. Eachspinal cord sample was cut into cervical, thoracic and lumbar sections.Each spinal cord section cut in half and both halves were placed inindividual cassettes per section (cervical, thoracic and lumbar) forprocessing. All three cassettes were embedded into one paraffin blockper animal. When applicable, portions of brain and spinal cord were snapfrozen and stored at −80° C.

hSMN-1 Western Blot Analysis

Standard western blot procedures were followed employing variousantibodies that recognizes hSMN protein, such as: (A) anti-SMN 4F11antibody at 1:1,000 dilution; (B) Pierce PA5-27309 a-SMN antibody at1:1,000 dilution; and (C) LSBio C138149 a-SMN antibody at 1:1,000dilution. For each experiment one microgram of hSMN mRNA was transfectedinto ˜1×10⁶ BHK-21 cells using Lipofectamine 2000. Cells were treatedwith OptiMem and harvested 16-18 hours post-transfection. Cell lysateswere harvested, processed and loaded on to an 8-16% Tris Glycine gel.The gel was transferred using a PVDF membrane and treated with therespective primary antibody. Goat anti-mouse HRP antibody was used asthe secondary antibody at 1:10,000 dilution for 45 minutes at roomtemperature followed by washing and development. The data demonstratesthat each antibody tested showed a strong signal for hSMN-1 and wasspecific for human SMN, as indicated by an absence in a cross-reactivesignal for untreated BHK cells (FIG. 11).

In Situ Hybridization (ISH) Analysis

Tissue from each representative sample, was assayed for hSMN-1 mRNAusing a manual in situ hybridization analysis, performed using RNAscope®(Advanced Cell Diagnostic) “ZZ” probe technology. Probes were generatedbased on the codon-optimized sequence of human SMN messenger RNA (SEQ IDNO: 4). Briefly, the RNAscope® assay is an in situ hybridization assaydesigned to visualize single RNA molecules per cell in formalin-fixed,paraffin-embedded (FFPE) tissue mounted on slides. Each embedded tissuesample was pretreated according to the manufacturers protocol andincubated with a target specific hSMN-1 RNA probe. The hSMN-1 probe wasshown to be specific for human SMN-1 and had little to no crossreactivity with mouse or rat SMN-1. Once bound, the hSMN-1 probe ishybridized to a cascade of signal amplification molecules, through aseries of 6 consecutive rounds of amplification. The sample was thentreated with an HRP-labeled probe specific to the signal amplificationcassette and assayed by chromatic visualization using3,3′-diaminobenzidine (DAB). A probe specific for Ubiquitin C was usedas the positive control. Positive SMN signal was compared to that ofuntreated and vehicle control treated rat or mouse tissue. Stainedsamples were visualized under a standard bright field microscope.

Immunohistochemical Analysis

Human SMN-1 mRNA-loaded lipid nanoparticles were administered to ratsvia intrathecal injection, and tissue samples collected and processed 24hours post administration in accordance with the methods describedabove. Rat spinal tissue samples were then assayed for hSMN-1 proteinexpression. Briefly, fixed tissue embedded in paraffin was processed andplaced on slides. The slides were dewaxed, rehydrated and antigenretrieval was performed using a pressure cooker with citrate buffer.Several blocking buffers were employed followed by primary antibodyincubation overnight at 4° C., using the 4F11 antibody at a 1:2500dilution. The resulting slides were washed and incubated at ambienttemperature with the secondary antibody polymer followed by washing andsubsequent chromagen development. The data demonstrates that in aslittle as 24 hours post intrathecal administration of hSMN-1 mRNA,staining is observed for human SMN-1 protein when compared tono-treatment control (FIG. 13). This supports the previous findingswhich demonstrate delivery of hSMN-1 mRNA to the spinal tissue.Furthermore, the data demonstrates that once delivered to the cellhSMN-1 mRNA is effectively expressed to generate hSMN-1 protein.

Results

The data presented in this example demonstrates that intrathecaladministration of hSMN-1 mRNA loaded liposomes (e.g., lipid orpolymer-based nanoparticles) results in successful intracellulardelivery of mRNA in neurons in the brain and spinal cord, includingthose difficult to treat cells, such as anterior horn cells and dorsalroot ganglia.

The results have shown that mRNA encapsulated within a lipidnanoparticle (e.g., lipid nanoparticle comprising cKK-E12) can beeffectively delivered to various tissues of the CNS followingintrathecal administrations. Using the exemplary formulation disclosedin Table 6, mRNA was effectively delivered and internalized withinvarious neurons of the spinal cord (FIGS. 12A-12C), as verified by insitu hybridization assay. Surprisingly, intracellular mRNA delivery wasdemonstrated in the difficult to reach neuronal cells of the anteriorhorn, located deep within the tissues of the spinal column (FIGS.12A-12C). Little to no background was observed with mouse or rat SMN-1,indicating specificity for the human SMN-1 probe. Positive SMN signalwas compared to that of untreated and vehicle control treated rat ormouse tissue. Stained samples were visualized under a standard brightfield microscope.

These data demonstrates that the lipid or polymer nanoparticle basedmRNA delivery approach described herein was able to successfullypermeate the complex and dense cell membrane of the spinal cord neuronsand deliver the mRNA payload for the production of encoded proteinsinside neurons. It was particularly surprising that the mRNA deliveryapproach described herein was equally successful in permeating difficultto treat neurons such as anterior horn cell and dorsal root ganglia.Thus, the data presented herein demonstrates that lipid or polymernanoparticles, such as those comprising cKK-E12, may serve as apromising option for delivering mRNA to neuronal cells in the treatmentof a CNS disease. In particular, the present example demonstrates thathSMN mRNA loaded nanoparticles can be effectively delivered to neurons,including those difficult to treat motor neurons in the spinal cord, andcan be used for the production of SMN protein and treatment of spinalmuscular atrophy.

Example 5. In Vivo CO-CFTR-C-His₁₀ mRNA Delivery to CFTR Knockout Mice

Messenger RNA Synthesis.

For the experiment, C-terminal His₁₀ tagged codon-optimized human cysticfibrosis transmembrane conductance regulator (CO-CFTR-C-His₁₀) (SEQ IDNO:8) (“His₁₀” disclosed as SEQ ID NO: 11) and non-taggedcodon-optimized human CFTR (CO-CFTR) (SEQ ID NO:9) mRNA were synthesizedby in vitro transcription from a plasmid DNA template using standardmethod. mRNAs used in this example and Example 6 were produced by IVT inwhich 25% of U residues were 2-thio-uridine and 25% of C residues were5-methylcytidine.

Analysis of Human CFTR Protein Produced Via Intratracheal AdministeredmRNA-Loaded Nanoparticles.

For the study, CFTR knockout mice were used. CFTR mRNA formulation orvehicle control was introduced using a PARI Boy jet nebulizer. Mice weresacrificed and perfused with saline, after a predetermined period oftime, to allow for protein expression from the mRNA.

PEI Formulation.

PEI formulation has been used to deliver CFTR mRNA to the lung and wasused as a control in this experiment. Polymeric nanoparticleformulations with 25 kDa branched PEI were prepared as follows. Therequired amount of mRNA was diluted just before application in water forinjection (Braun, Melsungen) to a total volume of 4 ml and added quicklyto 4 ml of an aqueous solution of branched PEI 25 kDa using a pipette atan N/P ratio of 10. The solution was mixed by pipetting up and down tentimes and nebulized as two separate 4.0 ml fractions one after anotherto the mouse lungs using the indicated nebulizer.

cKK-E12 Formulation.

For the lipid-based nanoparticle experiment, a lipid formulation wascreated using CO-CFTR-C-His₁₀ RNA in a formulation ofcKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 50:25:20:5 (mg:mg:mg:mg)).The solution was nebulized to the mouse lungs using the indicatednebulizer.

Nebulization (Aerosol) Administration of Human CO-CFTR-C-His₁₀ mRNA.

CFTR test materials were administered by a single aerosol inhalation viaPARI Boy jet nebulizer (nominal dose volume of up to 8 mL/group). Thetest material was delivered to a box containing the whole group ofanimals (n=4) and connected to oxygen flow and scavenger system.

Administration of Human CO-CFTR-C-His₁₀ mRNA.

CFTR mRNA was prepared in the manner described above. Four CFTR knockoutmice were placed in an aerosol chamber box and exposed to 2 mg totalcodon optimized unmodified human CFTR mRNA (comprising the codingsequence of SEQ ID NO: 8) via nebulization (Pari Boy jet nebulizer) overthe course of approximately one hour. Mice were sacrificed 24 hourspost-exposure.

Euthanasia.

Animals were euthanized by CO₂ asphyxiation at representative timespost-dose administration (+5%) followed by thoracotomy andexsanguinations. Whole blood (maximal obtainable volume) was collectedvia cardiac puncture and discarded.

Perfusion.

Following exsanguination, all animals underwent cardiac perfusion withsaline. In brief, whole body intracardiac perfusion was performed byinserting 23/21 gauge needle attached to 10 mL syringe containing salineset into the lumen of the left ventricle for perfusion. The right atriumwas incised to provide a drainage outlet for perfusate. Gentle andsteady pressure was applied to the plunger to perfuse the animal afterthe needle had been positioned in the heart. Adequate flow of theflushing solution was ensured when the exiting perfusate flows clear(free of visible blood) indicating that the flushing solution hassaturated the body and the procedure was complete.

Tissue Collection.

Following perfusion, all animals had their lungs (right and left)harvested. Both (right and left) lungs were snap frozen in liquidnitrogen and stored separately at nominally −70° C.

Expression of Human CFTR in CO-CFTR-C-his₁₀ in CFTR Knockout Mice.

CFTR expression was detected by Western blot analysis of tissue lysatecollected from CFTR mRNA-treated mouse lungs. Mature “C” band wasdetected in left and right lungs of all treated mice, for both thecKK-E12-based and PEI-based formulations (FIG. 14). Expression of themature “C” band was verified by comparison with lysate collected fromHEK 293T human CO-CFTR-C-His₁₀ positive cells. In contrast, nodetectable signal was observed in lysate collected from wild typeuntreated control mice (FIG. 14). Taken together, these data suggestthat cKK-E12 may be used to deliver mRNA (e.g., CFTR mRNA) to the lungvia, e.g., inhalation, as effectively as or even better than PEI basedformulations.

Example 6: In Vivo Expression in the Lung

This example further demonstrates successful in vivo expression in thelung following aerosol delivery of mRNA-loaded ckk-E12 basednanoparticles. All studies were performed using pigs of the GermanLandrace, obtained from Technical University Munich, Weihenstephan,Germany. The pigs had a body weight ranging from 35-90 kg.FFL/CO-CFTR-C-His10 mRNA formulation or vehicle control was introducedusing a Pari jet nebulizer. Pigs were sacrificed and perfused withsaline, after a predetermined period of time, to allow for proteinexpression from the mRNA.

Messenger RNA Synthesis.

In the example, codon optimized fire fly luciferase (CO-FFL) mRNA wassynthesized by in vitro transcription from plasmid DNA templates.

cKK-E12 Formulation.

For the lipid-based nanoparticle experiment, a lipid formulation wascreated using 1 mg FFL+9 mg of CO-CFTR-C-His₁₀ mRNA encapsulated in aformulation of cKK-E12:DOPE:Chol:PEGDMG2K (relative amounts 40:30:25:5(mol ratio). The solution was nebulized to the Pig lungs using theindicated nebulizer.

Aerosol Application.

The aerosol (Saline or CO-FFL cKK-E12 formulation) was nebulized andinhaled into the anaesthetized pig. Sedation in pigs was initiated bypremedication with azaperone 2 mg/kg body weight, ketamine 15 mg/kg bodyweight, atropine 0.1 mg/kg body weight and followed by insertion of anintravenous line to the lateral auricular vein. Pigs were anesthetizedby intravenous injection of propofol 3-5 mg/kg body weight as required.Anesthesia was maintained by isoflurane (2-3%) with 1% propofol bolusinjection at 4 to 8 mg/kg body weight to enhance anesthesia as required.Duration of the anesthesia was approximately 1-3 hrs. Pigs were killedwith bolus injection of pentobarbital (100 mg/kg body weight) andpotassium chloride via the lateral ear vein. Lungs were excised andtissue specimens were collected from various lung regions followed byincubation in cell culture medium overnight. The stored samples weresubjected to bioluminescence detection.

Bioluminescence Analysis.

For measurement of luciferase activity, tissue specimens were eitherhomogenized and analyzed in a tube luminometer or incubated in a mediumbath comprising D-Luciferin substrate and subjected to ex vivoluciferase BLI. The data illustrate that a strong bioluminescence signalwas observed for each of the (A)CO-FFL/CO-CFTR-C-His₁₀ mRNA treatedpigs, when compared to (B) control lung tissue samples from control pigs(Saline vehicle control) (FIGS. 15 A&B).

These data illustrate that FFL/CFTR mRNA were successfully delivered toand expressed in the lung by aerosol administration of a cKK-E12 basedlipid formulation.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1. A method of delivery of messenger RNA (mRNA) in vivo, comprisingadministering to a subject in need of delivery a composition comprisingan mRNA encoding a protein, encapsulated within a liposome such that theadministering of the composition results in the expression of theprotein encoded by the mRNA in vivo; wherein the liposome comprises acationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein: p is an integerof between 1 and 9, inclusive; each instance of R² is independentlyhydrogen or optionally substituted C₁₋₆ alkyl; each instance of R⁶ andR⁷ is independently a group of the formula (i), (ii), or (iii); Formulae(i), (ii), and (iii) are:

wherein: each instance of R′ is independently hydrogen or optionallysubstituted alkyl; X is O, S, or NR^(X), wherein R^(X) is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, or a nitrogen protecting group; Y is O, S, orNR^(Y), wherein R^(Y) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group; R^(P) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to a sulfur atom, or a nitrogenprotecting group when attached to a nitrogen atom; and R^(L) isoptionally substituted C₁₋₅₀ alkyl, optionally substituted C₂₋₅₀alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substitutedheteroC₁₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl,optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.
 2. The methodof claim 1, wherein the cationic lipid is cKK-E12:


3. The method of claim 1, wherein the liposome further comprises one ormore non-cationic lipids, one or more cholesterol-based lipids and/orone or more PEG-modified lipids.
 4. The method of claim 3, wherein theone or more non-cationic lipids are selected from DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)).
 5. The method ofclaim 3, wherein the one or more cholesterol-based lipids arecholesterol and/or PEGylated cholesterol.
 6. (canceled)
 7. The method ofclaim 3, wherein the liposome comprises cKK-E12, DOPE, cholesterol andDMG-PEG2K. 8-12. (canceled)
 13. The method claim 1, wherein the liposomehas a size less than about 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, or 50nm. 14-35. (canceled)
 36. A method of treating a disease or disordercomprising administering to subject in need of treatment a compositioncomprising an mRNA encoding a therapeutic protein encapsulated within aliposome such that the administering of the composition results in theexpression of the protein encoded by the mRNA in one or more tissuesaffected by the disease or disorder; wherein the liposome comprises acationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein: p is an integerof between 1 and 9, inclusive; each instance of R² is independentlyhydrogen or optionally substituted C₁₋₆ alkyl; each instance of R⁶ andR⁷ is independently a group of the formula (i), (ii), or (iii); Formulae(i), (ii), and (iii) are:

wherein: each instance of R′ is independently hydrogen or optionallysubstituted alkyl; X is O, S, or NR^(X), wherein R^(X) is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, or a nitrogen protecting group; Y is O, S, orNR^(Y), wherein R^(Y) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group; R^(P) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to a sulfur atom, or a nitrogenprotecting group when attached to a nitrogen atom; and R^(L) isoptionally substituted C₁₋₅₀ alkyl, optionally substituted C₂₋₅₀alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substitutedheteroC₁₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl,optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.
 37. The methodof claim 36, wherein the cationic lipid is cKK-E12:


38. A composition for delivery of messenger RNA (mRNA) comprising anmRNA encoding a protein encapsulated within a liposome, wherein theliposome comprises a cationic lipid of formula I-c:

or a pharmaceutically acceptable salt thereof, wherein: p is an integerof between 1 and 9, inclusive; each instance of R² is independentlyhydrogen or optionally substituted C₁₋₆ alkyl; each instance of R⁶ andR⁷ is independently a group of the formula (i), (ii), or (iii); Formulae(i), (ii), and (iii) are:

wherein: each instance of R′ is independently hydrogen or optionallysubstituted alkyl; X is O, S, or NR^(X), wherein R^(X) is hydrogen,optionally substituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl, optionallysubstituted heteroaryl, or a nitrogen protecting group; Y is O, S, orNR^(Y), wherein R^(Y) is hydrogen, optionally substituted alkyl,optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, or anitrogen protecting group; R^(P) is hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, optionally substituted heteroaryl, anoxygen protecting group when attached to an oxygen atom, a sulfurprotecting group when attached to a sulfur atom, or a nitrogenprotecting group when attached to a nitrogen atom; and R^(L) isoptionally substituted C₁₋₅₀ alkyl, optionally substituted C₂₋₅₀alkenyl, optionally substituted C₂₋₅₀ alkynyl, optionally substitutedheteroC₁₋₅₀ alkyl, optionally substituted heteroC₂₋₅₀ alkenyl,optionally substituted heteroC₂₋₅₀ alkynyl, or a polymer.
 39. Thecomposition of claim 38, wherein the cationic lipid is cKK-E12:


40. The composition of claim 39, wherein the liposome further comprisesone or more non-cationic lipids, one or more cholesterol-based lipidsand/or one or more PEG-modified lipids.
 41. The composition of claim 40,wherein the one or more non-cationic lipids are selected from DSPC(1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC(1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleyl-sn-glycero-3-phosphoethanolamine), DOPC(1,2-dioleyl-sn-glycero-3-phosphotidylcholine) DPPE(1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE(1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG(,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol)). 42-56. (canceled)